ANTIOXIDANTS & REDOX SIGNALING Volume 12, Number 11, 2010 C OMPREHENSIVE INVITED REVIEW © Mary Ann Liebert, Inc. DOI: 10.1089/ ars.2009.2698

Redox Regulation of DNA Repair: Implications for Human Health and Cancer Therapeutic Development

2 2 Meihua Luo~ Hongzhen He, Mark R. Ke l ley ~ -3 and Millie M. Georgiadis .4

Abstract

Red.ox reactions are known to regulate many important cellular processes. In this revievv, v.re focus on the role of redox regulation in DNA repair both in direct regulation of specific DNA repair proteins as well as indirect transcriptional regulation. A key player in the redox regulation of DNA repair is the base excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APEl) in its role as a redox factor. APEl is reduced by the general redox factor thioredoxin, and in turn reduces several important transcription factors that regulate expression of DNA repair proteins. Finally, we consider the potential for chemotherapeutic development through the modulation of APEl's redox activity and its impact on DNA repair. Antioxid. Redox Signal. 12, 1247-1269.

l. Introduction 1248 II. DNA-Repair Pathways 1248 A. Mammalian d irect repair: 0 6-alkylguanine-DNA methyltransferase or 0 6-methylguanine-DNA methyltransferase 1249 B. Base-excision repair 1249 C. Nucleotide-excision repair 1249 D. Mismatch repair 1250 E. Nonhomologous DNA end-joining and homologous recombiJ.1ation 1250 ITT. General Redox Systems 1251 A. The thioredoxin system 1251 B. The glutaredoxin/glutathione system 1252 C. l~oles of general redox systems 1252 N. The Redox Activity of APEl 1252 A. Evolution of the redox function of APEl 1252 B. Comparison of APEl with other redox factors 1254 C. Mechanism of redox regulation by APE1 1255 V. Transcription Factors Regulated by the Redox Activity of APEl 1255 A. p53 1256 B. AP-1 1257 C. HIF-la and hypoxia 1258 VT. The Multifunctional APEl and Redox Control 1259 VIL Modulating APEl Activities as a Cancer Therapeutic Approach 1260 A. APEl redox inhibitors 1260 1. E3330 1260 2. Other redox iJ.1hibitors 1261 B. APEl repair iJ.1hibitors 1261

Reviewing Editors: Margherita Bignami, Diego Bonatto, Dindial Ramotar, Young R. Seo, and Silvia Tomaletti

1Department of Pediatrics (Section of HematoloSj,'./ Oncology), Herman B. Wells Center for Pediatric Research, Indiana University; 2 Department of Biochentistry and Molecular Biology, Department of Pharmacology and Toxicology, Indiana U1tiversity School of Medicine; and 4 Department of Chemistry and Chemical Biology, Indiana University-Purdue University at Indianapolis, Indianapolis, lndiana.

1247 1248 LUO ET AL.

VIII. Chemoprevention, Redox Modulation, and DNA Repair 1261 A. Dietary antioxidants 1261 1. Ellagic acid 1261 2. Selenium 1261 3. Oltipraz 1262 B. Direct regulation of DNA repair by altered redox status of the cell 1262 IX. Concluding l~emarks 1262

I. Introduction overvievv of general redox systems as well as an in-depth discussion of the redox activity of APEl. Finally, in consider­ LTHOUGH THE IMPORTANCE of DNA-repair pathways in ing the impact of redox regulation of DNA repair to human A protecting the genome from damage caused by endog­ health, we discuss the modulation of the redox activity of enous and exogenous DNA-damaging agents (40, 44, 60) has APEl by small molecules and the potential for chemothera­ long been recognized, the role of redox regulation in these peutic development targeting redox regulation of DNA repair. pathways is a relatively recent discovery. In writing this re­ vie'~', vve attempted to guide the reader through general as II. DNA-Repair Pathways well as specific aspects of DNA repair and redox regulation, focusing ultimately on the connection beh-veen the h-vo. We The genome of eukaryotic cells is constantly under attack begin vvith an overview of DNA-repair pathways leading to a from both endogenous and exogenous DNA-damaging more in-depth discussion of one specific DNA-repair path­ agents. DNA damage resulting from endogenous agents in­ way, the base excision repair (BER) pathway. We focus on the cludes oxidation by reactive oxygen species (ROS) generated BER pathway, which is responsible for the repair of DNA from normal 1netabolic processes, alkylation by agents such as damage caused by oxidation, alkylation, and ionizing radia­ 5-adenosylmethionine, adduct fonnation resulting from at­ tion, and specifically on apurinic/apyrimidinic endonuclease 1 tack by reactive carbonyl species fonned during lipid perox­ (APEl ), the only DNA-repair protein currently known to serve idation, hydrolytic depurination leading to the formation of a dual role as a repair enzyme and a redox factor. In its role as a abasic sites, or deamination of bases, primarily cytidine, and redox factor, APEl modifies downstream trai1scription factors to a lesser extent, adenine (44). Exogenous agents include such as AP-1, NF-KB, CREB, p53, and others, and thereby in­ envirorunental insults (chemicals, carcinogens, UV light), directly alters the activity of other DNA-repair pathways. To che1notherapeutic agents, and radiation dan1age (40, 60). put the redox activity of APEl in perspective, we provide an Failure to repair DNA damage in both postmitotic and mitotic

Direct Base Mismatch Nucleotide Non-homologous Homologous Repair Excision Excision End Joining Recombination (HR) (NHEJ) ! MSH2/6 TCR GGR Glycosylase ~ MSH2/3 ! ! RNA Polll, DDB-XPE, Ku70,Ku80 APE1 CSA, CSB, XPC, XPAB2 HR2313 I Long Short MLH1-PMS2 Patch Patch MLH1 -PMS1 DNA-PK

RPA, XPA, XPC­ TFllH, XPB, XPC, XPD, XPG, XPF- ERCC1 MRN, Rad51, Rad52, Rad54, BRCA1, BRCA2, PCNA, PolS/&, ! Ligase I EX01, RFC, PCNA, RPA, RFC, PCNA, DNA Pol ~1. XRCC4, NA PolS, Ligase PolS/&, Ligase I Ligase 4, Artemis

FIG. 1. Schematic overview of DNA-repair pathway. Several DNA-repair pathways are involved in maintaining cell genomic stability; these include direct repair (DR), base-excision repair (BER), nucleotide-excision repair (NER), mismatch repair (MMI{), homologous recombination (HR), and nonhomologous end joining (NHEJ). More than 150 proteins are involved. Only selected genes of each path\·vay ai·e shown here. [Adapted from Fishel et al. (57).) 1249 REDOX REGULATION OF DNA REPAIR cells can result in apoptosis or accumulation of mutations and B. Base-excision repair even cell-cycle arrest (58, 106). For example, the DN~-dam~ge BER is responsible for the repair of DNA damage arising response in 1nitotic cells results in cell-eye!~ ar_rest mvolvmg fro1n alkylation, d eamination, or oxidation of bases (8, 40, 50). the major cell-cycle machinery. Tn postm1totic cells, DNA Alkylation of bases arises from exposi.rre to either endogenous d amage may result in cell-cycle activation and subsequent agents such as S-adenosylmethionine or exogen~us agents, arrest, leading to deleterious events in this cell population as including environmental and chemotherapeutic agents, well (110, 160). However, we have evolved a series of DNA­ w hereas deamination of cytidines and adenines occurs spon­ repair pathways to correct the damage, incl_uding ~~ect repa~ taneously. Oxidative damage can result from ROS generated (DR), base-excision repair (BER), nucleot1de-exas1on_ re~arr by normal cellular processes, in addition to envirorunental or (NER), mismatch repair (MMR), homologous recomb1n~ti on chemotherapeutic agents. BER is initiated by the removal of (HR), and nonhomologous end joining (NHEJ) (85, 86) (Fig.~) . the damaged base through enzymes called DNA glycosylases, The number of DNA-repair proteins and factors involved 111 which specifically recognize several different types of base the cellular response to DNA damage keeps growing as damage. Glycosylases are of two types, monofunctional and m ore and more information is obtained, not only on the DNA bifunctionaL Monofunctional glycosylases (e.g., N-methyl repair enzymes involved in each path,.vay, but also on the purine DNA glycosylase (MPG or AAG)] excise the d~a~ed regulatory networks that are induced by persis ~ence o_f DNA base to generate an apurinic/ apyrimidinic (AP) or abas1c site, darnage in the cell (182). Distinct DNA damage IS reparred by which is acted on by the multifunctional AP endonuclease, the different pathways and mechanisms. Overlap and inte_r­ APEl. Bi functional glycosylases such as human 8-oxoguanine action between the various pathways and some overlap In ONA glycosylase (hOGGl), human endonuclease VIII- like 6 mechanisn1s occur. For example, 0 -n1ethylguanine can DNA glycosylase (NEILl-3), and E. coli endonucl~ase III 6 be removed directly by 0 -m ethylguanine-DNA methyl­ (NTH) glycosylase have an additional A!' lyase function (~6, transferase (MGMT or AGl) in DR, but if this pathway is not 43) that excises the damaged base and rucks the phosphod1e­ successful, the 0 6mG mispairs and is recognized by the MMR ster backbone 3' to the AP site. The resulting AP site is pro­ pathway (59). Similarly, oxidative DNA dam~ge is ~epaired cessed by APE1, which hydrolyzes the phosphodiester mainly by BER, but som e repair by NER also lS possible (53). backbone immediately 5' to the AP site, creating 3' OH and 5' Single-sh·and DNA breaks (SSBs) unrepaired by BER lead to deoxyribose phosphate (5' dRP) termini. At this stage, repair d ouble-strand breaks (DSBs), which may be repaired by HR, can proceed by two pathways: the short-patch B El~ (SP-BEl~) and HR can also repair DNA DSBs that NHEJ pathways fa il to pathway and the long-patch BER (LP-BER)_ p~th.way. APEl is process (49). Interaction of different DNA-repair pathways responsible for 95°/o of the endonuclease act1v1ty 1n the cell and and mechanisms provides the most efficient defense for the is a critical part of both the short-patch and the long-patch BER cell genome, whereas reduced repair capacity ca:' lead . to pathway (45, 46). SP-BER repairs normal AP sites. DNA genomic instability. A number of diseases are as~oc1a ted with polymerase f3 (pol /3) removes the 5' dRP moiety by its dRPase defects in DNA repair, including xeroderma p1gmentosum , activity and u ses the 3' OH terminus to insert the correct b~s~ . Cockayne syndro1ne, tridothiodystrophy, Werner syndrome, The nick is ligated by DNA ligase ill/ XRCCl, and reparr lS and Bloom syndrome (118, 162). The reader is directed to completed. The LP-BER pathway preferentially repairs oxi­ recent comprehensive reviews for more specific information dized and reduced AP sites and is a minor branch of the BER on each DNA-repair pathway (47, 111, 144, 145, 150, 157, 174). pathway. A segment or fl ap of three to eight n_u c leo~des Sttr­ For updated information on the individual repair proteU:-S, the rounding the AP site is displaced, followed by insertion of t~e following link m ay prove i.1seful: http://W\.vw_.cga l.1cnet/ correct nucleotides by DNA polymerase b, s, or {3, along with DNA_Repair_Genes.html (182). What follows is an over­ proliferating cell nuclear antigen (PCNA) and replication view of ONA-repair pathways necessary to provide a con­ factor-C (RF-C). After resynthesis, flap endonuclease 1 (FENl) text for understanding the role of redox regulation in DNA removes the displaced strand, and DNA ligase T, or the DNA repair. ligase ill/ XRCCl complex ligates the nick. APEl i..r; the only AP endonuclease that performs these functions in the BER, and as A. Mammalian direct repair: d'-alkylguanine-DNA such, is a key player in the BER process. APEl also coordinates methyltransferase or d'-methylguanine-DNA recruitment of other DNA-repair proteins involved in BER throu gh a comp lex network of direct methyltransferase pr~tei~-pro tein inter~c­ tions and indirect interactions, as shown in Fig. 2. N o effective This type of repair in mammals is tern1ed direct reversal backup to APEl activity exists in the cell'. as is disc~. in because the damaged base is repaired through removal of the more detail later, including its other tna1or redox-s1gnaling alteration to the base instead of ren1oval of the d a1naged base. function. It is i.mique in this sense and probably is the most efficient mechanism of repair (105). The protein that carries out this C. Nucleotide-excision repair reaction, the AGT protein, removes alkyl groups through di­ rect transfer from the 0 6 position of guanine and to a lesser The NER pathway is responsible for repairing large ad­ extent from the 0 4 position from thymine to the protein, ducts such as ultraviolet-light- induced cyclobutane pyrimi­ leaving a guanine or thymine in DNA and ina c~va ted protein. d ine dimers, adducts induced by polycyclic aromatic This L5 a stoichiometric reaction, as one protem removes one hydrocarbons, and other bulky DNA lesions _induced. by alkyl group and is then degraded. It is essential to repair cross-linking agents and base-damaging che1m ca~ carci_no­ 0 6-meG adducts, as they cause errors by mispairing with gens. Numerous proteins are required to co~plet~ ~ ER: More thymine during replication, leading to G:C to A:T transitions than 25 proteins/ complexes have been identified m e~­ karyotic cells; these can be further divided into two main or a strand break. 1250 LUO ET AL.

YB-1 ,....~ XPG _.~~ ~ ... / WRN """"::::::::::------.._ ... ~ ____.....;::,,....,.....-- _____ ~ @ --- ~ =::::::::::------=:: ~ 1 SUM0-1/-3,.... /'1 I 1 DBP XPC~ 1 1 1 MSHS / I I I I I I I I I I I I I I @Y"

FIG. 2. Network of protein- protein interactions involving APEl (adapted from ref. 51). APEl plays an essential role in BER through its enzymatic activity as well as its role in coordinating interactions, either directly (solid lines) or indirectly (dashed lines), with a large nLnnber of other proteins involved in BER. Proteins highlighted are central components with APEl, apurinic/apyrimidinic endonudease, in the middle. Those proteins in the first column are DNA glycosylases; the remaining proteins include PCNA, an accessory protein; polymerases, pol Pand pol <5; ligase 1, LIGl; and the flap endonuclease, FENl.

subpathways; global genome repair (GGI~) and transcription­ ple, when MSH2 is paired with MSH6, it recognizes both coupled repair (TCR), depending on the complexes that ini­ insertion-deletion mispairs and single-base mismatches, tiate repair (8, 63). TCR is initiated ""hen RNA polymerase II whereas \·vhen it is paired with MSH3, the complex recog­ (RNA Pol TI) stalls at sites of DNA damage. TCR-specific nizes insertion-deletion mispairs. After recognition, MSH factors, including the Cockayne syndrome proteins, CSA and proteins recruit MLHl and its binding partners, post-meiotic­ CSB, are recruited at the site of transcription arrest, follo;ved segregation increased-I protein (PMSl) and PMS2. An by removal of the lesion by NER enzymes. In contrast, the exonuclease removes the DNA lesion, a DNA polymerase heterodirner XPC/ HR23B appears to be the major damage­ synthesizes a new strand, and finally, a DNA ligase completes recognition factor in hun1an cells. The UV-DNA damage­ the repair. This has been previously reviewed (104, 126). binding protein UV-DDB is additionally required for NER of UV-induced cyclobutane pyrimidine d imers. After recogni­ E. Nonhomologous DNA end-joining tion, both TCR and GGR use the same proteins to repair the and homologous recombination damaged DNA. The transcription-factor ITH (TFIIH) complex NHEJ is the main repair pathway for DSBs in mamma­ is recruited to the site of DNA damage, including its compo­ lian cellc;. DNA DSBs may be caused by ionizing radiation (IR), nent heli.cases, XPB and XPD (xerodem1a p igmentosum chemotherapeutic drugs, cleavage during V (D) J-recombi­ complementary group B and D proteins) that unwind the nation, meiotic recombi11ation, or the collapse of replication DNA strand on either side of the DNA damage. XPA and RPA forks. DSBs are the most severe fo1m of DNA dan1age and (replication protein A) stabilize the exposed single-strand endanger genomic stability by coordinating deletion or DNA followed by cleavage of the 27- to 30-nucleotide frag­ translocation or both of chromosomal DNA. Proteins in­ ment 3' and 5' of the lesion by endonucleases XPG and duding, but not limited to, Ku 70, Ku 80, DNA ligase IV, and ERCCl/XPFl/XPF. The resulting gap is filled in by the DNA XRCC4 are part of the NHEJ-repair pathway. The Ku proteins polymerases{> ore, along with PCNA, RPA, and replication bind to the ends of broken DNA and, as a complex with DNA­ factor C (RFC) by using the undamaged strand as a template. PKs (DNA-dependent kinase catalytic sub\.nut), interact '~1 ith DNA li.gase IV ai1d XRCC4 to repair DNA through the NHEJ D. Mismatch repair pathway. DNA ligase IV and XRCC4 function in a complex to In a broad definition, MMR is responsible for the recogni­ ligate the nick and to complete repair (34). Discovery of neV\' tion and repair of single mismatches or misaligned short proteins involved in NHEJ includes Metnase or SETMAR nucleotide repeats. Mismatches can be endogenously caused (117), which has been shown to interact with DNA ligase lV by spontaneous deamination of 5-methylcytosine to thy1nine, and to enhance the efficiency and accuracy of NHEJ (92). resulting in a guanine-to-thyinine mismatch, damage to the Homologous recombination (HR) also is i11volved in re­ cellular nucleotide pool, cytosine deamination to uracil, pairing DNA DSBs. HR is initiated through the DSB recog­ resulting in a guanine-to-uracil mismatch, or incorrect incor­ nition by ATM (ataxia telangiectasia-mutated protein), which poration by DNA polymerase. A complex of MSH2 and MSH6 phosphorylates multiple downstream proteins. DSBs are recognizes the mismatd1 and initiates the pathway. Various processed by the MRN complex (Mrell/Rad50/Nbsl) nu­ conwi.nations of MSH2 and either MSH3 or MSH6 are fom1ed, clease activity to yield single-sh·and DNA (ssDNA). ATM­ which specify the type of mismatch recognized. For exam- activated BRCAl attracts BRCA2 and RADSl to bind to the REDOX REGULATION OF DNA REPAIR 1251

H •.. SH ·-----.. s /s -~ /s TRX: I")>rot -TRX, / Prot -TRX, ""-SH S / ""-SH ···S/ ""-s

Mixed disulfide intermediate

FIG. 3. Reduction of oxidized proteins by TRX. Thioredoxin (TRX) reduces oxidized proteins containing a disulfide through the formation of a mixed disulfide intermediate involving nucleophilic attack by Cys 32 of the CXXC motif. The mixed disulfide is then resolved by Cys 35, resulting in the formation of a disulfide bond in thioredoxin. Ribbon renderings are shown for the reduced (PDB identifier, lEl{T) and oxidized thioredoxin (PDB identifier, lEl{U) along with the Cys residues of the CXXC motif in black-stick renderings.

ssDNA ends, allowing the RAD52/ RAD54 complex to join reduced state) (69, 100). Cys can exist in a number of different and fom1 larger co1nplexes with BLM and WRN proteins. forms in vivo, including cysteinyl radical, sulfenic acid, sulfi­ These large protein complexes at the strand break direct nic acid, sulfonic acid, cystine, and others [see Jacob et al. (100) pairing of the processed DNA with a homologous region on for a recent review of sulfur chen1istry associated with Cys]. the sister chromatid and initiate strand exchange. This was Of the types of reactions involving Cys residues, the thiol/ previously reviewed in detail (167). disulfide exchange reaction Le; of relevance to our dLc;cussion of general redox systems [reviewed in (69)]. In thiol/ disulfide­ Ill. General Redox Systems exchange reactions, an oxidized protein, including a disulfide bond, is recognized by a reduced protein such as TRX or In living systems, two systems are primarily responsible for GRX/ GSH and is then reduced through the formation of an general reduction-oxidation (redox) regulation, the thior­ intermediate mixed disulfide bond (Fig. 3). Cys acts as a edoxin (TRX) and glutaredoxin/ glutathione (GRX/ GSH) nucleophile in this type of reaction (69, 100). Interestingly, systems. They maintain the redox cellular homeostasis as '~' ell Tl{)( and GRX/ GSH are reduced by thioredoxin reductase as redox regulate several cellular processes through a thiol­ and glutathione reductase, respectively, in a reaction involv­ redox mechanic;m (91, 142). Thiol-based redox mechanisms ing both thiol/ disulfide exchange and electron-transfer reac­ rely on the special properties of Cys residues, which can adopt tions requiring cofactors such as FADH and NADPH (Fig. 4) 10 different sulfur oxidation states from +6 to -2 (the fully 2 (69, 100)

S TR SeH HS TR Se A. The thioredoxin system - XI:< I HS/ 'S Components of the thioredoxin system include thioredoxin k~sH T TRX-(SH2) (TRX), NADPH, and thioredoxin reductase (TR) (90, 100). FADH2 FADH2 Thioredoxins (TRXs) comprise a large family of structurally 1 Xs conserved proteins that serve as general protein disulfide TRX/ I HS TR SeH HS TR eH °""s oxidoreductases and can reduce disulfide bonds in a va1iety of proteins through a thiol/ disulfide exchange mechanism H;>=p< SH HS SH (143). Oxidized thioredoxin is then reduced by thioredoxin

FAD FADH2 reductase, a flavoprotein containing a selenocysteine, in a reaction involving NADPH. K Thioredoxins (Tl{)(s) share a similar active-site motif Cys­ NADPH + H• NADP• X-X-Cys and a common structural motif, kno,vn as the TRX fold (91, 120, 153), 'vhich consists of a four-stranded /J-sheet FIG. 4. Thioredoxin reductase/ thioredoxin (TR/ TRX) surrounded by three a-helices (Fig. 4). The active-site motif is redox cascade. Thioredoxin is reduced by thioredoxin located on the loop connecting /J-sheet 1 and a-helix 1. The N­ reductase in a somewhat more-complex mechanism involv­ ing the formation of a selenylsulfide and subsequent reduc­ terminal Cys residue in the active site is surface exposed and tion by a pair of Cys residues within another subunit of TR. has a lo,.v pK. value; for example, Cys32 in human TRX has an estimated pK. of 6.3 (61), whereas the C- terminal Cys is Electron-transfer reactions involving the FADH 2, a cofactor of Tl{, and NADPH are required to regenerate TR. [Adapted buried in the molecule and has a much higher pK., value. It has fro1n Jacob et al. (100).] been proposed that the low pK., value of the N-terminal Cys 1252 LUO ET AL. arises from the partial positive charge from the dipole cellular redox state, which is an important metabolic variable, mon1ent associated with et.-helix 2 (88), or alternatively, may influencing many aspects of cell function, like growth, apo­ be due to its hydrogen bond to the C-term.inal Cys (181). The ptosis, and reductive biosynthesis. Tn addition, by redox sig­ nucleophilicity of the thiolate group of the Cys is increased by naling, they control the activation of a number of transcrip tion the low pK• . The proposed reaction mechanic;m of disulfide factors and hence regulate a broad range of cellular functions reduction by thioredoxin is as follows: the N-term.inal cyste­ (11, 153, 192). The two redox systems physiologically play ine thiolate of TRX acts as a nucleophile and attacks the target many roles in different organisms and, conversely, are also disulfide, resulting in a transient mixed disulfide inte1me­ pathophysiologic factors for a va1iety of hun1an diseases, in­ diate, vvhich is, in turn, reduced by the C-te1minal active­ cluding cancer, viral disease, Alzheimer's disease, and others site Cys residue, generating a dithiol in the target protein (7, 12, 33, 154), and hence serve as vital drug targets for cancer and a disulfide in thioredoxin (91, 108, 120) (Fig. 3). The therapy and other disease treatments (20, 39, 122, 123, 141). resulting disulfide in the active site of TRX can be reduced by Although the thioredoxin system and glutaredoxin syste1n Tl~ with electrons from NADPH, completing the catalytic share a number of ftu1ctions, they are not just simple duplicate cycle. systems; TRX and GRX act on different substrates (54, 121). The mechanism by ,.vhich TR reduces TRX back to the di­ TRX but not GRX, for example, has been implicated in the thiol involves the fonnation of a selenylsulfide in the active reducti on of APE1 (6, 83, 94, 170). site of TR (100), as shown in Fig. 4. A second redox active site The remainder of our discussion on redox factors focuses located in the other subunit of the dimeric TI~ contains two on APE1, which is the only DNA-repair protein kno,-vn also thiols that reduce the selenylsulfide back to a thiol and selenol, to have a role in redox regulation affecting the expression of a with the resultant formation of a disulfide bond. This dis­ number of other DNA-repair proteins. ulfide is reduced by electron transfer from FADH21 and the resulting FAD is then reduced by electron transfer from IV. The Redox Activity of APE1 NADPH (100). In a search to identify the nuclear factor responsible for B. The glutaredoxin/ glutathione system reducing the transcription factor AP-1., a factor termed redox effector factor 1, Ref-1, '~' as identified (184, 187). Since this The glutaredoxin system is composed of NADPH, the initial dic;covery, APEl (Ref-1) has been reported to reduce a flavoprotein glutathione reductase, glutathione, and glutar­ number of other ilnportant transcription factors, includil1g edoxin (54, 90, 121). This system also works through a cas­ NF-KB, HIF-la, p53, PAX, and others (35, 48, 83, 84, 112, 169, cade of disulfide oxidation and reduction. Glutaredoxins 175) (Fig. 6). And, as discussed below, the redox activity of (GRXs) are small redox enzymes of ~ 100 amino acid resi­ APEl plays an important role in regulating the expression of a dues, which use glutathione as a cofactor. Structurally glu­ large number of DNA-repair proteins. taredoxins are very similar to thioredoxins, retaining the same fold and active sites. However, the active site of GRXs A. Evolution of the redox function of APE1 includes Cys-X-X-Cys or Cys-X-X-Ser. By using a similar reactive mechanism, GRXs catalyze the reversible reduc­ Although APE1 is reported to have distinct redox and re­ tion of substrate protein disulfides, resulting in oxidation of pair domains (186) located "vithin the N- and C-term.inal re­ the GRXs (Fig. 5). Oxidized GRXs are reduced none­ gions of the protein, respectively, these functional domains do nzymatically by glutathione (glutamyl-cysteinyl-gly­ not correspond to independently folded domains within the cine,GSH), and then the oxidized glutathionine disulfide proteil1 (i.e., structural domains). Furthermore, the repair and (GSSG) is reduced by glutathione reductase at the expense of redox activities do not appear to be coordinated within hu­ NADPH (121). man APEl, and '"'hereas the AP endonuclease activity of APEl is conserved from bacteria to humans, the redox func­ tion is unique to mammals. Tl1us, as shown in Fig. 7, APEl C. Roles of general redox systems and E. coli exonuclease lil, the major AP endonuclease found Tlu·ough a thiol/disulfide exchange mechanism, thior­ within E.coli, are closely related in terms of shucture (r.m.s.d., edoxin and glutaredoxin systems maintain a reducing intra- 1.5 A), retaining not only the sa1ne overall fold and topology

H. FIG. S. Reduction of oxidized proteins SH ...... S S ___::;S by glutaredoxin/ glutathione (GRX/ H GSH ). Through a mechanism similar to GRX /~ I "'/ Prot - GRX / ""Prot rot that used by TRX, GRX also forms a SH S .-··S/ H mixed-disulfide intennediate '"'ith an oxidized protein. This disulfide is re­ Mixed disulfide intermediate solved through involvement of a second Cys residue of the CXXC motif in GRX, resulting in reduction of the protein and GSH s- sG GSH formation of a disulfide bond in GRX. GRX/ GSH directly reduces GRX again through - a disulfide-exchange mechanism and is ~SH itself reduced by glutathione reductase. [Adapted from Lillig et al. (121).] REDOX REGULATION OF DNA REPAIR 1253

Redox Regulation DNA Repair

APE1/Ref-1 redox activity inhibited _ ._G-A-A-T-C ­ -~C-T-~A-G- AP-1 ! p53 Glycosylase (Ogg1 , Nth1) NF kB -...... -r-c- (reduced) (oxidized) HLF Binding of -~o-r-r-A-~ ---- HIF-1cx APE1 to the Methoxyamine APE1/Ref-1 ~ CREB AP site ATF blocked. _ T-G * "A-:::- ~...!.( .:...M.:...X:-.) __. (oxidized) (reduced) Egr-1 - A-C-T-T-~ - NF-Y l PAX CRT0044876 Redox control of Transcription factors

APE1/Ref-1 ---• l repair activity inhibited - A-C-T-T-A-G­ Gene expression changes ONA Ligase - T-C r#&i- C- - P-{;-T-T-A-G-

FIG. 6. APEl has dual roles in redox and DNA repair. APEl possesses two major functions: redox regulatory/ signaling and DNA repair. Tirrough its redox function, APEl regulates gene expression by modifying the redox status of some tran­ scription factors involved in variety of cancer processes. S1nall molecules that block APEl redox function are shown in ovals. In addition to its redox function, APEl plays a critical role in the BER DNA-repair pathway as an AP endonuclease, which processes the AP sites. Blocking AP sites by using methoxyamine (MX) or APEl or both directly by using APEl-specific inhibitors such as CRT0044876 may decrease DNA repair and lead to tumor-cell death. [Adapted from Luo et al. (128).)

but also very similar endonudease active sites (Fig. 8). The sequence identity between APEl and exonuclease III is 27.7%. The most obvious structural difference between human APEl and exonuclease III is an additional 62 N-terminal residues found only in APEl (Fig. 7). Within this N-terminal region of APEl is a nuclear localization sequence. However, addition of N-tenninal residues alone does not confer redox activity; zebra fish APE includes a similar N-terminal addition (Fig. 9) but lacks redox activity (68). So the question then becomes, what is required for the re­ dox activity of human APEl (hAPEl)? This continues to be a source of controversy in the literature. Of the seven Cys res­ idues present in hAPEl, Cys 65 1.vas identified as the critical residue required for redox activity tlu·ough analysis of single Cys-to-Ala substitutions within APEl (see Fig. 10) (178). In­ vestigation of the role of Cys residues \.vithin APEl was based N-terminal------region of hAPE1 on the finding that a Cys residue within the DNA-binding domain of the transcription factor c-Jun was subject to oxi­ FIG. 7. Comparison of human APEl and exonuclease III dation, leading to loss of DNA bindings and \.vas reduced by from Escherichia coli. The structurally similar enzymes, APEl (2, 184, 187). Subsequently, the crystal structure of human APEl (PDB identifier, 1 BIX, green ribbon rendering) human APEl was reported (70), revealing that Cys 65, a res­ and exonuclease III (PDB identifier, lAKO, blue), share a similar fold; however, APEl includes an additional 62 N­ idue unique to mammalian sequences, is a buried residue tenninal residues, highlighted in red (residues 44-62). In located on the first fJ strand in the fold, which is part of a fJ exonuclease TIT, the residue equivalent to the APE1 Cys 65 sheet in the core of the protein (Fig. 10). The residue equiva­ (yellmv stick rendering) L5 Val 4 (gray stick rendering). (For lent to the hAPEl Cys 65 in exonuclease III, based on struc­ interpretation of the references to color in this figure legend, tural aligrunent, is Val 4, whereas that in zebrafish APEl the reader is referred to the web version of this article at (zApe) is Thr 58 (68). Conservation of Cys residues beh.veen WW\-v.l iebertonline.com/ ars). 1254 LUO ET AL.

FIG. 8. Compar ison of active-site regions of human APEl and exonuclease III. Stick renderings are shown for residues within the active sites of APEl (PDB identifier, lBIX, green ribbon rendering) and exonuclease III (PDB identifier, lAKO, blue). Active-site residues are color coded as follows: light blue, Asn; slate blue, Gln; blue, Asp; purple blue, Glu; red, His; green, Tyr; light orange, Phe; bright orange, Trp; yellow, Leu; pale yellow, Ile. The active-site regions of APEl and exonu­ FIG. 10. Positions of Cys residues within APEl. The clease III are highly conserved. (For interpretation of the re­ human APEl (PDB identifier, lBIX, gray ribbon rendering) ferences to color in this figure legend, the reader is referred to includes seven Cys residues (black sticks), whose positions the web version of this article at www.liebertonline.com/ ars). relative to the active-site His 309 are sho~vn. None of the Cys residues is appropriately positioned to form a disulfide the hAPEl and the E. coli enzyme is llinited to Cys 208 and bond, and the redox-critical Cys residue, Cys 65, is a buried Cys 310, but within vertebrate APEs, all Cys residues except residue located in the first P strand of the APEl fold. Cys 65 and Cys138 are conserved (68). The structure of APE in vertebrates also is conserved, based on a comparison of the 58, the structure and chemical environment is highly con­ zAPE and human APEl structures. Within the vicinity of Thr served (68). In contrast, residues in the vicinity of Val 4 in the E. coli enzyme exonuclease TIT are not similar to those found in hAPE1 N-terminus the vertebrate APEs. The report of a viable C64A knockin mouse and data \

B. Comparison of APE1 with other redox factors ln contrast to molecules such as TRX and GIV<, which FIG. 9. Comparison of zebrafish APE and human APEl. maintain the general redox status of the cell, APEI does not Although the zebra fish APE (PDB identifier, 203C, light gray) contain t'~' O Cys residues within a C-X-X-C motif. Thus, the also includes an extended N-terminus similar to that found mechanism by which APE1 reduces transcription factors is in the human APEl (PDB identifier, 203H, dark gray), it does likely to differ from that of thioredoxin or glutaredoxins. In not have redox activity. As sho~vn in these ribbon render­ ings, the zebrafish and human enzymes are structurally very the crystal structures reported to date of APEI, no disulfide similar, including the N-terminal residues. Conserved Cys bonds are present, and the only Cys residue reported to be residues, including 93, 99, 208, 296, and 310, are shown in absolutely required for redox activity is Cys 65, which is a black stick rende1ings for the human enzyme. buried residue. The Cys residues positioned closest to one REDOX REGULATION OF DNA REPAIR 1255

Local unfolding of C-terminus in oxidized PRX C-terminal-- region in reduced PRX "'...

FIG. 11. Redox mechanism of peroxiredoxin (PRX) involves local unfolding. Peroxiredoxin is the enzyme responsible for detoxification of hydrogen peroxide. In a mechanism dissimilar to that used by TRX or GRX, PRX has two Cys residues, one located in each subunit of the dimeric structure, which participate in the reduction of hydrogen peroxide, leading to the for1nation of disulfide bond in PRX. Reduced PRXII (PDB identifier, lQMV) is shown in the left panel as a ribbon rendering with one subunit in light gray, the second in dark gray, and Cys residues in black. Tn the right panel, the oxidized PRXI (PDB identifier, 1QQ2) is rendered similarly. Unfolding of the C-te:minus of the dark-gray subunit allows the formation of a disulfide bond between the Cys residues that are located ~9 A apart in the reduced form of the protein.

another are 93 and 208, but their respective S atoms are ~3 .5 peroxiredoxin is the proximity of a C ys residue to a terminus; A apart, too far apart to fom1 a disulfide bond, which is typ­ in the case of hAPEl, Cys 65 is located relatively close to the ically ~2.2 A in length. Further, these residues are buried in N-terminus of the protein; it is located \~rithin the first sec­ the core of the protein and are not accessible. The solvent­ ondary stn1ctural element (a /3 strand) in the fold (Fig. 7). Tn accessible Cys residues include 99 and 138; however, these summary, we conclude that hAPEl is unique as a redox fac­ residues are not in close proximity to one another and would tor, having evolved this additional function while maintain­ not be expected to interact to form a disulfide bond. Fur­ ing its essential base-excision repair activity. the1more, substitution of Ala for either Cys 99or138 has no effect on redox activity (178). Before the determination of the C. Mechanism of redox regulation by APE1 crystal structure of APEl, it was proposed that Cys 65 and 93 Although a role for Cys 65 in the redox activity of hAPEl form a disulfide bond (178). Given the respective locations of has been established (68, 178), a detailed mec11anismhas yet to Cys 65 and Cys 93 in the protein, > 8 A• apart and positioned be elucidated. Jt is possible that another as-yet-unidentified on opposite sides of the {3 sheet (Fig. 10), a substantial con­ Cys residue in APEl is involved in the redox activity. In this formational change in the structure of the protein would be case, after the formation of a mixed disulfide intermediate required for a disulfide bond to form between these residues. beh.veen Al)El and a transcription factor, a resolving Cys Another group of redox proteins, peroxiredoxins, are re­ would serve to restore the thiolates in the transcription factor. sponsible for sensing hydrogen peroxide in the cell and serve As noted earlier, this interaction would likely involve a dif­ as catalysts to detoxify this extremely reactive molecule (87). ferent conformation of hAPE1 than has been reported in the These enzymes are dissimilar to thioredoxin- or glutaredoxin­ crystal structures to date (17, 68, 70, 139). Alternatively, a type molecules in that they lack a C-X-X-C n1otif, but they do residue other than Cys might be involved in the redox activ­ include two Cys residues that are required for activity (183). ity, perhaps a Ser residue, as found in some glutaredoxins. A These Cys residues are located ~9 A from one another in the similar mechanism would be proposed in this case, although fully folded dimeric stn1cture (Fig. 11); one Cys from each involvement of a Ser residue ;vould require a significant re­ monomer forms the active site (183). The nucleophilic thiolate duction in its pK•. As the stoichiometry of the relevant redox is sequestered before a local unfolding event near the dimer complex has yet to be established, it is possible that more than interface. The resolving thiolate is located near the C-terminus one APEl molecule is involved in the reduction of transcrip­ of the molecule and, on local unfolding, is placed in close tion factors. In this case, a Cys or Ser from a second molecule proximity to the nucleophilic thiolate (183) (Fig. 11 ). Peroxir­ of APEl may serve as the resolving thiolate, again, in a edoxins reduce hydrogen peroxide and not other proteins mechanisn1 similar to that proposed for thioredoxin. As this (87), but on overoxidation to the sulfenic or sulfinic acid state, problem is of considerable interest, we are actively investi­ are themselves reduced by sulfiredoxin (29, 177). The re­ gating the mechanism of redox regulation by hAPE1. quirement for local unfolding for peroxiredoxin to complete its catalytic cycle in the detoxification of hydrogen peroxide is V. Transcription Factors Regulated very interesting and may be a general mechanism used by by the Redox Activity of APE1 other redox factors. Although APEl does not include two Cys residues positioned similarly to those found in peroxiredoxin, Because APEl is a multifunctional protein involved in both the possibility remains that a conformational change or local the repair of DNA damaged by oxidative or alkylating com­ unfolding event may result in more favorable positioning of poimds and in the redox regulation of a nurnber of stress­ Cys residues. An interesting similarity bet\~reen APEl and inducible h·anscription factors, such as AP-1, NF-KB, HIF-lo:, 1256 LUO ET AL.

A B pathways (80, 162). Regulation of DNA repair by p53 is complex, involving both transactivation-dependent and -in­ dependent mechanisms, revie,~red by Sengt1pta and Harris (162), and p53 is itself regulated by both redox-dependent and -independent mechanisms involving Ref-1 (APEl). lt1vestigation of redox regulation of p53 was initiated based on the finding that oxidized p53 bound DNA very poorly (75) and led to the discovery that Ref-1 was the factor responsible for enhancing the DNA-binding activity of '.vild-type p53 (101 ). Although Ref-1 is believed to interact transiently '.vith p53, and this interaction has been detected only by far Wes­ tern analysis (67) and not by coimmunoprecipitation (101), c D Ref-1 stimulates DNA-binding activity in vitro and transacti­ vation in vivo by wild-type p53 (67, 101). The mechanis1n by which Ref-1 enhances the DNA-binding activity of p53 has not yet been fully elucidated but has been proposed to include redox-dependent activation of the DNA-binding domain, potentially through direct reduction of a disulfide bond, as well as a redox-independent interaction with the C-terminal regulatory domain of p53 (101). Most recently, Ref-1 \·vas sho,.vn to interact '.vith the tetramerization domain, promot­ ing formation of tetramers from dimers and thereby enhanc­ FIG. 12. Structural comparison of DNA-binding domains ing sequence-specific DNA-binding activity of p53 through a from transcription factors that are reported to be redox redox-independent mecha11ism (77). l~edox signaling involv­ regulated by APEl/Ref-1 bound to DNA. Proteins are ing p53 has also been shown to depend on a general redox shov.rn in a gray ribbon rendering, and the DNA in a spline/ factor as well as Ref-1. TRX has been shown to enhance stick representation in black. The DNA-binding domains of stimulation of p53-dependent expression of p21 by Ref-1 the leucine zipper-motif protein AP-1 (c-Jw1/c-Fos) bound to suggesting a link between TRX and Ref-1 in cellular response DNA (PDB identifier lFOS) is shown in (A), the immuno­ to oxidative stress (175). A summary of p53 regulation of DR, globulin-like fold of p53 (PDB identifier 1TSR) with an a BER, NER, MMR, HR, and NHEJ repair pathways follows. helix bound in the major groove (B), and the heterodinieric two-domain imn1unoglobulin-like folds of p65/ p50 NF-KB BER is initiated by highly specialized DNA glycosylases (PDB identifier 1VKX) in (C). A representative basic leucine­ that cleave the DNA base, creating an AP site, as discussed zipper/ basic helix-loop-helix-containing protein (MyoD) earlier. Regtilation of the BER path,

TABLE 1. !~OLE OF APEl IN REGULATING EXPRESSION OF DNA REPAIR GENES Apel/Ref-1 Transcription Factor

NF kB AP-1 CREB p53 HJF-la Direct Repair Direct Repair ACT ACT Hornologous Recombination Homologous l~econibination Honzologous Recombination A1M RADSl NBSl RADSO WRN Global Geno1ne Repair Global Genome Repair ERCCl Xl'C XPA DDB2 RAD23B ERCC3 Misrnatch I

DNA BER process between a nurnber of transcription factors Finally, some studies in murine fibroblast cell lines have that are under redox control and the DNA-repair response demonstrated that AGT also is under p53 regulation (72, 155). (Table 1). ACT also appears to be regulated by NF-KB, another tran­ NER, which is divided into TCR and CCR, as discussed scription factor that is under redox control by APEl. This was earlier, is affected differentially by p53. For example, several demonstrated by overexpression of the p65 subunit of NF-KB studies fotu1d that p53 selectively affected GGR, but not TCR. in HEK293 cells, resulting in an increase in AGT expression T\·vo main proteins in GGR, DDB2 and XPC, "''hich are in­ (116). volved in DNA-damage recognition, are transcriptionally All of these studies point to the possible relevance of redox regulated by p53 (3, 96, 168). Loss of p53 and subsequent controlling DNA-repair responses through p53. Therefore, if deficiencies in the CCR proteins DDB2 and XPC appear to reduced p53 is required to bind DNA and either activate or lead to genome instability. This has been effectively demon­ repress the transcription of DNA-repair genes, as discussed strated in knockout mouse studies. In this study, lOOo/o of earlier, then it follo>vs that Ref-1 (APEI) plays an i.J.nportant xpc·I· mice develop lung cancer, and DDB2·/· mice develop role not only in the redox modulation of p53 but also in the skin tumors (89). Again, if p53 is not fully functional through regulation of DNA repair. redox modification, it cannot turn on DDB2 and XPC, which would result in defective CGR (Table 1). B. AP-1 MMI~, which is in charge of DNA repair after DNA polymerase errors, removes mismatches in DNA. MSH2 in Activator protein-I (AP-1) refers to a family of structurally complex '~1ith MSH6 or MSH3 is active in the recognition of and functionally related basic leucine zipper proteins (bZIPs) single-base mismatches and short insertion/deletion mispairs that intermix to form heterodimeric sequence-specific DNA­ or larger loops of unpaired nucleotides, respectively. MSH2, binding proteins, including primarily Jun proteins, c-Jnn, MLHI, and PMS2 have all been sho\.vn to be under p53 reg­ JunB, and JnnD, Fos proteins, c-Fos, FosB, Fra-1 and Fra-2, ulation, similar to DDB2 and XPC in NER (38, 159). PCNA, and some ATP-family members, ATFa, ATF-2, and ATF-3 another member of the NER pathway, also is under p53 reg­ (82). These proteins recognize AP-1 sites that are also refen·ed ulation (Table 1) (189). to as tetradecanoylphorbol-13-acetate {TPA)-responsive ele­ DSBs threaten severely genomic stability by facilitating ments. AP-1 transcription factors are inducible factors that deletion or translocation or both of chromosomal DNA. Be­ respond to environmental changes, including stress and ra­ cause either a deficit or an excess in Hl~ may lead to genomic diation, or to growth-factors signals. Proliferation, differenti­ instability, it is not surprising that HR is highly regulated. ation, apoptosis, and transformation are some of the processes Once again, p53 plays an important role in the repair of DSBs that are mediated by AP-1 (82). through the regulation of both DSB repair path\o\rays, HR and The first report of red ox activity associated with APE1 and NHEJ. Increased levels of HR have been observed in mice that its identification as Ref-1 resulted from efforts to identify the are deficient in wild-type p53 (26, 127). Arias-Lopez et al. (10) nuclear factor responsible for reducing AP-1 (c-Jun/c-Fos) and demonstrated that p53 inhibits HR through repression of thereby enhancing its DNA-binding activity (1, 184). l{edox RAD51 expression. Additionally, p53 has been demonstrated regulation of AP-1 was found to result from oxidation/ to repress the h·anscription of the RecQ4 helicases, WRN and reduction of conserved Cys residues within the basic DNA­ RecQ4 (163, 191). binding domains of c-Jun and c-Fos (AP-1) (2). Oxidized AP-l 1258 LUO ET AL. shows little affinity for DNA as compared with reduced AP-1. subunits (93) that plays an important role under hypoxic c-Fos and c-Jun di.Jnerize through a coiled-coil interaction conditions in the cell. Of fue two subunits, HIF-lCt. has been involving the leucine-zipper motifs and bind DNA as a het­ sho,.vn to be crucial for regulating cellular response to hypoxia erodimer; c-Jun also forms a homodimeric species with lo,.ver and is frequently overexpressed in ht1man cancers. Under DNA-binding affinity than the heterodimeric c-Jun/ c-Fos, normal oxygen conditions, HIF-la is targeted for ubiquitin­ whereas c-Fos does not exhibit any DNA-binding activity on mediated proteasomal degradation by von Rippel-Landau its O\.Vn (2). The conserved C ys residue within c-Jun is mutated protein (pVHL) (97, 98, 132). Under hypoxic conditions, HIF­ to a Ser residue in the oncogenic v-Jun, ""hich is constitutively la translocates to fue nucleus and dimerizes with HIF-1/J, active and exhibits enhanced DNA-binding relative to c-Jun forming HlF-1. HTF-1, along '"'ith coactivators, binds hypoxia­ (2). Thus, Ref-1 regulates DNA binding of AP-1 in a redox­ response elements (HREs) within promoters and regulates the dependent manner involving direct reduction of the con­ expression of its downstream genes, including vascular en­ served Cys residues within the DNA-bi11ding domams of dothelial growfu factor (VEGF) (62, 102). The DNA-bindi11g c-Jun and c-Fos. l~ef-1 has been shown to copurify wifu AP-1 activity of HIF-1 has been shown to be regulated by redox (184) and thus interacts more stably ""ith this transcription signali11g, and the redox-dependent stabilization of the HIF- factor than '.vith p53. TRX has also been identified as a factor 1a protein is required for activation of HIF-1 (93). Over­ modulating transcriptional activation of AP-1 through its re­ expression of TRX and Ref-1 enhanced the DNA-binding duction of Ref-1 (83). As sttmmarized later, AP-1, which is activity of HJF-1, as detected in a reporter assay. The results, rapidly i11duced in response to a number of cellular stimuli, taken togefuer, suggest a role for I~ef-1 as a redox regulator of regulates fue expression of several protems involved pri­ HIF-la. l~ef-1 (APE-1) also has been shown to be required for marily in NER and MMR DNA-repail· pathways. the binding of transc1iptional protei11s to fue HIF-1 DNA­ Tn one example of regulation of DNA-repair proteins by recognition element within the rat pulmonary artery endo­ AP-1 composed of c-Jun and ATF2, Hayakav.ra et al. (78) thelial cell VEGF gene (198). Alfuough thic; study did not identified 23 DNA-repair or repair-associated genes whose specifically address the role of redox regulation, fue authors promoters are bound on phosphorylation of ATF2 and c-Jun found fuat l{ef-1 was required for the formation of the hyp­ after cisplatin treatment. These genes were identified by usi11g oxia-inducible transc1iptional complex, includi11g HIF-1 and chromatin inununoprecipitation (ChIP) vvifu antibodies transcriptional coactivators, p300 and cyclic AMP response against A TF2 and c-Jun, follo,~red by hybridization to pro­ element-binding protein (CREB). Tn a second example of Ref- moter arrays (78). These include ERCCl, ERCC3, XPA, MSH2, 1 as a coactivator, a transcriptional complex including HIP-la, MSH6, RAD50, RAD23B, MLHl, PMS2, UNG2, and ATM. signal and transducer of transcription 3 (ST AT3), CREB­ Confirmatory studies directly established expression of some binding protein (CBP)/ p300, and l~ef-1 (APEl) was reported genes, including ERCCl, ERCC3, XP A, RAD23B, and MSH2, to regulate the protein tyrosine kinase Src-dependent ex­ and so1ne genes that have been specifically in1plicated in fue pression of VEGF in response to hypoxia in pancreatic and repair of DNA-cisplatin adducts, such as RAD23B, XPA, prostate carcinomas (71). More recently, HTF-1Ct. was shown ERCC3, XPF-ERCCl, MSH2, and PMS2. DNA adducts in­ to play a role in downregulating mRNA and protein levels of duced by dsplatin are repaired mainly by fue NER pafuway. I~ef-1 w1der hypoxic conditions in human microvascular en­ Several important proteins including XI'A, I~D23B, El~CCl, dofuelial cells (125). Thus, HIF-lo: regulates expression levels and ERCC3 in NER were observed on the promoter array. The of Ref-1 and is itself regulated by Ref-1. members of the MMR complex, including MSH2, MSH6, Increasing evidence reveals fuat hypoxic stress in fue tumor MLHl, and PMS2, which are included in a large complex microenvironment can cause genetic instability in cancer cells. involved in the recognition of DNA-cic;platin adducts, are Hypoxia induces changes in the expression of several genes bound strongly by activated c-Jun or ATF2 or bofu. AP-1 also involved in DNA-repair pathways. These shtdies suggest that was discovered to upregulate MSH2 expression i.11 the mye­ hypoxia downregulates the expression of key genes '"'ithin loid leukeinia U937 cell line treated with a phorbol ester (TPA) fue MMR pathway, including MLHl and MSH2, and several (95). Undeniably, ERCCl has been recognized to be regulated critical mediators of HR, BRCAl, BRCA2, and RAD51, re­ by AP-1. Li et al. (119) demonstrated that AP-l is transcrip­ sulting in significant genetic instability (21, 22, 24, 25, 32, 109, tionally up-regulated ERCC-1 in response to TPA in human 135, 138) [reviewed in Bindra et al., 2007(23), and Bristow and ovarian cancer cells (119). Hill, 2008 (32)]. The MMR genes appear to be repressed All of fuese studies support fue role of fue involveinent of through a mechanism involvi11g c-Myc (25). AP-1 in regulating a significant nu1nber of DNA-repair pro­ In most studies, repression of MMR and HR has been teins fuat are involved mainly in fue NER and MMR path­ sho•vn to be independent of HIF-lo: (21, 22, 24, 25, 32, 135, ways (Table 1). Because AP-1 must be converted from an 138). However, this has been contradicted by Koshiji et al. oxidized to a reduced state to bind to its target sequence, (109), who demonstrated that HJF-la is responsible for genetic redox control of thic; protein would have significant implica­ instability during hypoxia at the nucleotide level by inhibiting tions. As for p53, APEl is implicated as a potential poi11t of MSH2 and MSH6, which recognize DNA base mismatches. control for regulating the DNA-binding activity of AP-1 and These investigators demonsh·ated fuat HIF-lc.< displaces fue thereby n1odulating the expression of DNA-repair protems. transcriptional activator Myc fro1n Spl binding to repress MSH2-MSH6 in a p53-dependent manner (109). HTF-1a also has been shown to be associated with the loss of MSH2 ex­ C. HIF-1CJ. and hypoxia pression in human sporadic colon cancers (172), linking Hypoxia-i11ducible factor - 1 (HJF-1) is a heterodirneric hypoxia to DNA repair in fue induction of these cancers. The transcription factor con1p1isrng HIF-la and HIF-1/J (also decrease of the expression of NBSl, a member of fue MRell­ known as aryl hydrocarbon-receptor nuclear h·anslocator) RAD50-NBS1 (MRN) complex that illitially recognizes DNA REDOX REGULATION OF DNA REPAIR 1259

DSBs, has also been shown to be HlF-la dependent (173). tissue-specific transcription factors (i.e., PEBP-2, Pax-5 and These studies dei11onstrate that the regulation of DNA repair -8, and TIF-1) (Fig. 6) (6, 35, 48, 84, 94, 112, 169, 175, 184, 185). is an integral part of the hypoxic response and that hypoxia ROS indticed by different oxidative or toxic agents have affects DNA repair partially through HTF-1a, a critical medi­ been shown to increase APE1 expression transiently (Fig. 13). ator in the hypoxic response (Table 1). As Ref-1 has been A number of transcription factors, including Egr-1, CREB, and implicated in the redox regulation of HIF-la, it then aLc;o plays Jun/ ATF4, are involved in the inducible expression of APEl a role in the regulation of DNA-repair genes controlled by (66, 73, 152). In a recent study, the level of APEl was shown to HIF-l o: . be increased h·anscriptionally in response to ROS in mela­ noma cells by microphthalmia-associated transcription factor (MiTF), a key transcription factor for melanocyte lineage VI. The Multifunctional APE1 and Redox Control survival that plays an important role in development and APEl is a vital multifunctional protein that acts as an carcinogenesis (122). In this study, melanoma cells were essential master regulator contributing to the maintenance of classified into MiTF-positive and -negative groups to explore the geno1ne stability (Fig. 6). Functional activities associated the function of MiTF in regulating cellular responses to ROS. with APE1 include apu1inic/ apyrimidinic endonuclease ac­ A high level of APE1 / Ref-1 was discovered in the MiTF­ tivity essential for BER, redox activity, transcriptional regu­ positive melanoma cell lines. Knocking down MiTF reduced latory activity (19), and most recently, RNA- cleavage activity the APEl protein level and abolished induction of APEl by (15, 176). In this review, we have limited our discussion to the l{OS. MiTF-negative melanoma cells survived more poorly APEl BER repair and redox activities, the functions relevant under ROS stress than did the MiTF-positive cells. Over­ to redox regulation of DNA repair. APEl also is subject to a expression of APEl partially rescued ROS-induced cell death nttmber of interesting posttranslational modifications, in­ when MiTF '.vas depleted. The MiTF regulation of APE1 is cluding acetylation, phosphorylation, and nitrosylation. The direct through E-boxes on the APEl promoter. It alc;o was implications of these modifications \.Vere reviewed recently found that exposure of HeLa cellc; to H2 0 2 and to hic;tone (19, 171) and are not further discussed here. deacetylase inhibitors increases acetylation of APEl at resi­ APEl has a pleiotropic role in controlling cellular response dues Lys6/ Lys7, leading to Egr-1-mediated induction of the to oxidative stress. In addition to its repair function in BER as tumor-suppressor PTEN gene expression (52). In addition, an AP endonuclease, APE1 controls the redox status of either increasing evidence demonstrates that functional triggering ubiquitous (i.e., AP-1, Egr-1, NF-KB, p53, CREB, HIF-lo:) or of membrane-bound receptors (such as TSH, CD40L, ATP,

Stimulation and Stress

NADH/NAOPHr-.-;;;;=-== ­ Oxldaset

TRX Kinases

ERK1/2

APE1/Ref-1

FIG. 13. Overview of ROS signaling. Reactive oxygen species (ROS) are generated through external stimuli or stress, as well as n1etabolic processes, and act as a signal for )NK and ERKl/ 2 kinases, resulting in the activation of a number of transcription factors, including Egr-1, CREB, and Jun/ ATF4, which are involved in the inducible expression of APEl. In melanoma cells, MiTF also was shown to upregulate transcription of APE1. In its role as a redox factor, APEl reduces a number of transcription factors, including AP-1, Egr-1, NF-KB, p53, CREB, and HJF-la. Thus, APEl controls the redox status of several transcription factors that in turn regulate expression of APEl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at w\~1 w.liebertonline.com/ ars). 1260 LUO ET AL.

IL-2) can lead to Al)El functional activation through intra­ dox function of Al'El and has no effect on APEl-repair ac­ cellular generation of sublethal doses of ROS (170). APEl also tivity and other members of the BER pathway (128). By using is directly responsible for the conh·ol of the intracellular ROS £3330, we demonstrated that APEl is required in normal levels through inhibiting the ubiquitous sma II GTPase Rael, embryonic hematopoiesis and that the redox function, but the regulatory subunit of NADPH oxidase system (9, 149). not repair activity, of APEl is critical in normal embryonic Recently, Park et al. (151) found that overexpressing APEl/ hematopoietic development (199). I~ef-1 increased inhibition of angiotensin Il (Ang II) to the 2 whole-cell conductance Ca - -activated K+ (BKca) currents in VII. Modulating APE1 Activities human umbilical vein endothelial cells (HUVECs) through as a Cancer Therapeutic Approach blocking NADPH oxidase-dependent ROS production. The inhibitory effect of Ang II on BKca channel function is Elevated APEl levels have been demonstrated in a variety NADPH oxidase dependent (151). It also was demonstrated of cancers and are typically associated with aggressive pro­ that NADPH-mediated ROS production induced by the P2Y liferation, increased resistance to therapeutic agents, and poor purinergic receptor triggering was able to promote APEl prognosis (SO, 140, 180, 190). Previous studies demonstrated h.1nctional activation (152) and results in the proposition of an that decreasing APEl / Ref-1 levels leads to the blockage of autoregulatory loop between these tl.vo systems. cell growth and the increase of cellular sensitivity to DNA­ We demonstrated that reducing expression of APEl in damage agents by using anti-sense oligonucleotides and neuronal cultures by using small interfering l~A (siRNA) sil~NA of APE1 / l~ef-1 (56, 58, 115, 147, 180). Not yet known is enhances cisplatin-induced ROS generation, cell killing, and the relative importance of APEl redox versus the repair apoptosis (103, 136). Another recent study showed that Apel function in cancer. Effo1ts to determine the effects of inhibiting can antagonize the generation of ROS. Overexpression of either the redox or repair function of APEl are ongoing. APEl inhibits, ""hereas silencing APEl expression potentiates Targeting of a specific protein, particularly one that plays an ROS accumulation under treatment with oxidative reagents important role in cellular response to stress, by chemical or loading with granzyme K in cytotoxic T lymphocyte knockout (i.e., through use of a small-molecule inhibitor) may (CTL)/ natural killer (NK) cells. APEl is a physiological sub­ have unintended consequences. However, exploration of sh·ate of granzyme K, and cleavage by granzyme K facilitates novel targets is clearly ai1 avenue that n1erits pursuit, partic­ inb·acellular ROS accumulation and enhances granzyme ularly in the case of cancers for which current treatments are K-induced cell death (74). Merluzzi et al. (136) also fotmd that ineffective. There has been considerable debate in the litera­ repression of APEl by antisense overexpression determines ture regarding the wisdom of inhibiting essential DNA-repair an additional increase in CD40-mediated B-cell proliferation, enzymes (14, 42, 57, 106, 129). However, small-molecule in­ and the increase is abolished by pretreatment of cells with the hibitors have been identified for several DNA-repair en­ antioxidant N-acetyl-L-cysteine (NAC). They proposed that zyines, including MGMT, poly ADP-ribose polymerase APEl, through control of the intracellular redox state, may (PARP1), ataxia-telangiectasia mutated kinase (A TM kinase), also affect the cell cycle by inducing nucleus-cytoplasm re­ APEl, and DNA PKcs (42, 57, 129). Targeting of the redox distribution of p21 (136). function of Al)El represents a novel approach iI1 the devel­ A genetic study has demonstrated that APEl is essential for opment of a cancer therapeutic agent. Blocking of the redox cell survival and organism development. Knockout of APEl function of APEl would be expected to affect the activity of a in mice leads to postimplantation embryonic lethality on days number of downstream transcription factors and the gene ES to E9 (188). Conditional knockout and knockdown strate­ products that they regulate. In this section, we focus primarily gies also confirmed the crucial role of this protein in cellc; (58, on redox inhibitors, those kno\.vn specifically to inhibit APEl 65, 99). In addition, studies demonstrated that altering APEl and those that may affect APEl either directly or indirectly. levels leads to a change of cell growth, survival, and sensi­ We briefly discuss Al'El-repair iilhibitors as they have been tivity (28, 37, 56, 58, 81, 115, 134, 147, 179, 180). Hovvever, reviewed recently (106). these studies used either overexpression of APEl, APEl an­ tisense oligonucleotides, or APE1 siRNA to change the total amount of cellular APEl and thereby all functions of APEl, A. APE1 redox inhibitors including its repair and redox activities. Thus, the function of 1. E3330. One molecule previously discussed '.vas APEl involved in each case of altered cellular function caimot demonstrated to biI1d specifically to APEl with a biI1ding be identified. Because APEl plays co1nplex and c1itical roles in constant esti.Inated by surface plasmon resonance analysis cell survival, proliferation, differentiation, and apoptosis in (SPR) of 1.6 nM, which suggests a specific interaction betl.veen physiologic and pathologic conditions, as '~'ell as in the APEl and E3330 (166). We recently dem.onstrated that E3330 gro~rth and development of the organic;m, it is important to blocks the redox h.mcti.on of APEl with AP-1 as the down­ distinguish and characterize which function of APEl is in­ stream target in vitro, as well as after the treatment of ovarian volved in different biologic events, especially those that may cancer cells with E3330 (128). Additionally, we found that differ in no1mal cells and various pathologic cells, such as £3330 blocks APEl redox activity ~1 ith HIF-let. and other cancer cells. The use of specific small-molecule inhibitors downstream transcription factors (Table 1 ). This demon­ blocking either repair or redox, but not both functions of strates that the redox inhibition is not specific for the do,.vn­ APEl, ,.vi.II give a clearer picture and will be helpful for stream target. Although E3330 blocked the redox function of modifying its ftmcti.on in treatment of the different diseases. APEl, it had no effect on APEl-repair endonuclease activity. Our recent data demonstrated that £3330, 2E-3-[5-(2, 3 di­ We also found that £3330 does have single-agent cancer cell­ methoxy-6-methyl-1,4-benzoquinolyl)]-2-nonyl-2-propenoic killing abilities in a variety of cancer cell lines representing acid]), a novel quinone derivative, specifically blocks the re- ova1ian, colon, lung, breast, brain, pancreatic, prostate, and REDOX REGULATION OF DNA REPAIR 1261 multiple myeloma cancers, but does not have significant cell number of recent reviews discuss the status of these types of killing in our studies with normal cells, such as emb1yonic agents (14, 57, 106). hematopoiesis cells, retinal vascular endothelial cells (RVECs), and human CD34+ progenitor cells. These data VIII. Chemoprevention, Redox Modulation, implicate the redox role of APE in cancer, but not in "normal" and DNA Repair cell survival. Inhibition of the APEl redox function signifi­ cantly attenuates I~VEC proliferation and capillary formation As was clearly documented, DNA dainage leading to in vitro but does not cause cell death. Furthermore, the capil­ genome instability is a key step for the initiation and pro­ lary formation of RVECs appears n1uch more sensitive to re­ gression of cancer (16). Both endogenous and exogenous dox inhibition of APEl than to the proliferation. This is the DNA-damaging agents and particularly those that induce first time that this role of APEl has been clearly demonstrated. oxidative stress are some of the main factors that cause DNA Additionally, our data demonstrate a new role of APEl in damage in cells. Therefore, agents that reduce the oxidative angiogenesis, and inhibition of APEl redox function by E3330 stress and subsequent DNA damage, as >veil as those that abrogates this role (128). E3330 also has been shown to inhibit increase the repair of DNA damage, are considered to be the gro,~r th of pancreatic cancer cell lines, an effect that is pertinent in cancer prevention. It is estimated that nearly one enhanced under hypoxic conditions (200), as well as pancre­ third of all cancer deaths in the United States could be pre­ atic cancer-associated endothelial and endothelial progenitor vented. Accumulating research evidence has shown that some cells (201). Consistent with redox regulation by l~ef-1, the dietary antioxidants are able to reduce the incidence of cancer DNA-binding activity of HIF-1 is inhibited by £3330 in the by increasing DNA repair and reducing oxidative stress. aforementioned pancreatic cancer studies. TI1us, collectively our data and those of others suggest that APEl redox function A. Dietary antioxidants will be a promising target of cancer treatment and will open a 1. Ellagic acid. Ellagic acid, a con1ponent in berries new avenue for cancer treatment. (bh.ieberry, strawberry, and red raspberry), was reported to reduce oxidative DNA damage both in vitro and in vivo (4, 5). 2. Other redox inhibitors. A number of natural products In vitro, ellagic acid has demonstrated a >95°/o inhibition of reported to affect either directly or indirectly the redox func­ 8-oxodeoxyguosine (8-oxodG) production. It also was shown tion of APEl in cells were recently revie~red (57, 107, 128, 133) to reduce other oxidative DNA adducts caused by 4-hydroxy- and are therefore discussed only briefly here. Soy isoflavones, 17/j-esh·adiol and CuC1 . In an in vivo study, female CD-1 mice a component in soybeans, are thought to have potential as 2 were fed pure ellagic acid, and formation of DNA adducts chemopreventive agents in prostate cancer (137). Treatment of was related to ellagic acid in a dose-dependent manner. Fur­ PC-3 prostate cells and xenograft mice with soy isoflavones ther srudy found both ellagic acid and its narural source after radiation treabnent resulted in increased cell killing, resulted in overexpression of genes involved in DNA-repair reduced NF-KB binding to DNA, and reduced APEl levels. pathways such as XPA, ERCC5, and DNA ligase III, mainly The authors concluded that the soy isoflavones reduced APEl those involved in NER. TI1ese results demonsh·ated that el­ levels and subsequently resulted in a reduction of the ability lagic acid is effective in preventing oxidative DNA damage of Al'El to reduce NF-KB, resulting in the inability of the cells both in vitro and in vivo by increasing DNA repair. to respond to the stress (156). Ho>vever, at this point, the data are merely co1Telative. Another nah1ral product, resveratrol, a component of red 2. Selenium. Selenium is found in plentiful amounts in wine and grapes, was reported to affect the redox activity of dairy, eggs, fish, meat, grains, and Brazil nuts. Selenium, in APEl (193). Resveratrol was shown to inhibit both the endo­ the form of selenocysteil1e, is a major constituent of many nuclease activity of APEl and the DNA-binding activities of antioxidai1t enzymes known as selenoproteins. Not surpris­ AP-1 in cellular extracts, presumably through inhibition of ingly, Se was reported to be preventive for cancer initiation APEl redox function. However, this has not been corrobo­ frorn oxidative DNA damage through reducing oxidative rated by others, nor has it been shown to be effective at levels stress and increasing DNA repair. The active species of Se that are physiologically relevant. include hydrogen selenide (H2Se) and its methylated metab­ olite, methylselenol (MeSeH), selenomethionine (SeMet), and selenoproteil1s. Se, in the form of selenomethionine, was re­ B. APE1 repair inhibitors ported to promote BER activity by p53 activation in normal To date, t>vo different classes of small molecules have been human fibroblasts in vitro (164). This sh1dy demonstrated that reported to inhibit the AP endonuclease activity of APEl, Se-induced p53 activation promotes BER activity by reducing methoxyarnine (18, 124) and negatively charged molecules specific cysteine residues in p53. A dominant-negative APEl including CRT0044876 (130), an aryl stibonic acid 13755 redox mutant blocks reductive activation of p53 by Se. Se also (161), and a number of pharmacophore-based compounds was shown to stin1ulate the activity of a selenoprotein, (197). Methoxyamine (MX) blocks repair by reacting with thioredoxin reductase (TR) (165). These data suggest that Se apurinic/ apyrirnidinic sites in the DNA and fom1ing stable reduces p53 through interactions involving TR, which re­ adducts that prevent endonucleolytic cleavage by APEl (18, duces TRX and APEl, as well as redox interactions between 64, 124). As MX acts at the level of the DNA, it would also be APEl and p53. Se-induced activation of p53 is also dependent expected to block the activity of other DNA-repair enzymes on the BI~CAl protein in recombinational repair, which is such as DNA polymerase f3 activity. However, none of the frequently mutated in fainilial breast cancer (55). It also was aforementioned co1npounds has been repo1ted to inhibit the reported that Se inhibited DNA-binding activity of tran­ redox activity of APEl; this is not discussed further here. A scription factors such as AP-1, NF-KB, SP-1, and SP-3, as '-veil 1262 LUO ET AL. as the DNA-repair proteins XPA in the NEl~ pathway and ti.on of OGGl activity by cadmium was strictly associated formamidopyrimidine DNA glycosylase (FPG) in the BER with reversible oxidation of the protein, as demonstrated by pathway (27, 76, 131, 195). TI1ese data i.J.nply that Se 1nay re­ the use of cysteine-modifying agents, such as diamide, 1-vith duce cancer incidence through modulating DNA repair, re­ the pure protein and cn1de extracts (30). A frequently found dox status of cells, and the cellular transcriptional response to polymorphism of OGG!, S326C OGGl, associated with can­ oxidative stress. It also suggests that the redox function of cer development, has been shown to have lower 8-oxoG DNA APEl is a major component of this interaction. Additional glycosylase activity, and the lower activity of OGG1-Cys326 is clinical studies have found an inverse relation beh~reen the associated '~'ith the easy oxidation of Cys326 to form a dis­ levels of Se and the prevalence of several types of cancer. ulfide bond (31). Tn this study, the 8-oxoG repair activity 1-vas analyzed in the cells and cell extracts of lymphoblastoid cell lines established from individuals carrying either Ser/Ser or 3. Oltipraz. Oltipraz (4-methyl-5-(pyrazinyl)-3H-l,2- Cys/ Cys genotypes. The cells homozygous for the Cys variant dithiole-3-thione), a synthetic dithiolethione, is similar to the display increased genetic instability, reduced 8-oxoG repair thiolethione, an antioxidant component in cruciferous vege­ rates, and almost twofold lower basal 8-oxoG DNA glycosy­ tables. Oltipraz has been shown to inhibit the development lase activity in their cell extracts. Reducing agents increase the and progression of multiple organ tumors, includi.J.1g breast, repair capacity to the level of the Ser va1iant, but do not affect bladder, colon, sto1nach, liver, lymph nodes, h.mg, pancreas, the activity of the latter. The 8-oxoG DNA glycosylase activity and skin, induced by a variety of stiucturally di.verse carcin­ in cells carryi.J.1g the Cys/ Cys alleles is more sensitive to oxi­ ogens in preclinical studies (41 ). Clinical studies demon­ dizing agents (31). strated that oltipraz has minimal toxicity in humans and NER, which repairs mainly bulky DNA adducts and heli.x­ significant chemopreventive activity (41). Oltipraz increased distorting lesions, has some crossover activity on oxidative NEI~ activity by decreasing platinum-DNA adducts, but not DNA damage. On exposure of human pulmonary epithe­ BER activity, as 1neasured by determining the levels of AP lial cell<> (A549) to nontoxic doses of H 0 , increased expres­ sites in HT29 colon adenocarcinoma cells (146). Further study 2 2 sion of XP A, XPC, ERCC4, and EJ~CCS was observed, whereas found that oltipraz also increases APEl protein level and AP-1 ERCCl expression decreased (114). Functional studies also DNA-binding, ;vhich is partially dependent on APEl in HT29 demonsti·ated a decrease in NER activity. cells (194). Oltipraz also was shown to inhibit microvessel Glutathione (GSH) also is directly implicated in the regu­ formation in both human and rodent bioassays in a dose­ lation of NER. GSH-depletion in cells preincubated with BSO, dependent manner (158). These data suggest that the redox, L-buthionine-sulfoximine, completely abolished the down­ not the repair, function of APE1 may be involved in chemo­ regulation of ERCC1 expression and the decrease in NER ca­ preventive effect of oltipraz. pacity by H 0 and increased significantly the upregulation All of these compounds are natural agents that have been 2 2 of ERCC4 expression. These data suggest that NER capacity as i.J.nplicated in protecting against cancer. well as the expression of NER-related genes can be modulated by oxidative stress (114). B. Direct regulation of DNA repair by altered redox status of the cell IX. Concluding Remarks

The evolutionarily conserved enzyme glyceraldehyde-3- Tn conclusion, redox regulation clearly plays an impor­ phosphate dehydrogenase (GAPDH) is a key redox-sensitive tant role in DNA repair, with implications for human health protein with an active-site cysteine sulfhydryl. GAPDH was and cancer therapeutic development. We have highlighted shown to interact directly with APEl (13). By using recombi­ the role of APEl, an important DNA-repair protein and nant protei.J.lS, Azam et al. (13) found thatGAPDH interacts with redox factor, in this revie\·v as a protei.J.1 directly linking APEl through the active-site cysteine 152 to convert the redox regulation and DNA repair. Tt is our expectation that "oxidized" form of APEl to its reduced form and reestablishes future research in this area ~ri ll bring additional insights the ability to cleave AP sites. This reduction process also en­ into the i.J.nportance of redox regulation in DNA repair and hanced the detection of APEl by anti-APE! antibodies, sug­ will likely result in the identification of other proteins that gesting a structural d1ange. siRNA knockdown of GAPDH in also play i.J.nportant roles i.J.1 redox regulation and DNA HCT116 cells enhanced sensitivity to the alkylating DNA­ repair. dan1aging agent methyl methane sulfonate (.MMS), v.rhich produces AP site, and ina·eased the level of spontaneous AP Acknowledgments sites in the genomic DNA. These data imply that GAPDH plays Financial support for this work was provided by the an i.J.nportant role in promoting BER activity by maintaining National Institutes of Health, National Cancer Institute APEl, a key AP endonudease, in an active reduced state. CA114571 to M.M.G., CA94025, CA106298, CA114571 and OGGl, an 8-oxoG DNA glycosylase in the BEI~ pathway, is CAJ21168 to M.R.K., TU Simon Cancer Center Translational responsible for recognizing and repairing 8-oxoguanine initiative pilot funding (!TRAC) to M.M.G. and M.R.K., and (8-oxoG), a com1non and mutagenic fo1m of oxidized guanine the Riley Children's Foundation (M.R.K.). in DNA. A recent study 1-vith human lymphoblastoid cells treated with cadmium resulted in an almost complete re­ duction in the 8-oxoG DNA glycosylase activity of OGGl, References presumably because of an alteration in the redox status of the 1. Abate C, Luk D, Gentz R, Rauscher FJ 3rd, and Curran T. cells. OGGl activity returned to normal once the redox cel­ Expression and purification of the leucine zipper and lular status was returned to normal. The reversible inactiva- DNA-binding domains of Fos and Jun: both Fos and Jun REDOX REGULATION OF DNA REPAIR 1263

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Abbreviations Used MPG = N-methylpurine DNA glycosylase MRN = Mrell/Rad50/ Nbsl 8-oxoG = 8-oxoguanine MSH2 = MutS homologue 2 AAG = alkyladenine DNA glycosylase MSH3 = MutS homologue 3 AGT = 0-6-alkylguanine-DNA methyltransferase MSH6 = MutS homologue 6 Ang II = angiotensin II MYH= MutY homologue AP- apurinic/ apyrimidinic NAC = N-acetyl-L-cysteine AP-1 = activator protein 1 NEIL = fgp/nei family DNA glycosylase APEl = apurinic/apyrimidinic endonuclease 1 NER = nucleotide-excision repair ATF4 = activating transcription factor 4 NF-KB = nuclear factor kappa B A TM= ataxia-tclangicctasia- mutated protein NHEJ = nonhon1ologous end joining BER = base-excision repair NK= natural killer BKca = large-conductance Ca2+ -activated K~ channels NTH= ho1nologue of Escherichia coli endonuclease BLM - Bloom syndrome gene product III (nth) BRCAl = breast cancer 1, early onset 06-meG =Cl' m.ethyl guanine BRCA2 - breast cancer 2, early onset P2Y = purinergic receptor CD40L - CD40 ligand PCNA = proliferating cell nuclear antigen . CREB = cyclic AMP response elen1ent-binding protein PMSl = postmeiotic-segregation increased-1 prote~ CSA = Cockayne syndrome protein A PMS2 = postmeiotic-segregation increased-2 protein CSB = Cockayne syndrome protein B Pol f3 = DNA polymerase /3 CTL = cytotoxic T ly1nphocyte PRX = peroxiredoxin DDBl = DNA damage binding protein 1 PTEN = phosphatase and tensin homologue deleted DDB2 = DNA damage binding protein 2 on chromosome 10 DNA-PI