THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 27, Issue of July 7, pp. 15915-15918, 1995 Minireview © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. damage in 12-13-nt-Iong oligomers and eucaryotes excise 27-29­ Excision Repair in nt-long fragments. This dual incision activity is referred to as Mammalian Cells* excision nuclease (excinuclease), The single-stranded gap gener­ ated by either type of excision is filled in by DNA polymerases and sealed by ligase. Aziz Saneart From the Department ofBiochemistry and Biophysics, Genetics ofExcision Repair University ofNorth Carolina School ofMedicine, The excision repair genes (uvrA, uvrB, and uvrC) of E. coli show Chapel Hill, North Carolina 27599 no homology to the human excision repair genes (1). In contrast, the sequences of excision repair genes in mammalian cells and There are two types of structural anomalies that lead to muta­ yeast are highly homologous, and the enzymology of excision repair tion, a permanent change in DNA sequence. The first class involves in these two systems is very similar (1, 3). Only mammalian exci­ normal bases in abnormal sequence context (mismatch, bulge, sion repair will be covered in this review. Three human diseases loop). The second class, which is referred to as DNA damage or are caused by a defect in excision repair (9): xeroderma pigmento­ DNA lesion, involves abnormal nucleotides (modified, fragmented, sum, Cockayne's syndrome, and trichothiodystrophy. cross-linked) in normal sequence context. DNA lesions, in addition Xeroderma pigmentosum patients suffer from photosensitivity, to causing mutations, also constitute replication and transcription photodermatoses including skin cancers, and in some cases from neurological abnormalities. XP patients are defective in excision blocks. repair. Mutations in 7 genes, XPA through XPG, cause XP. In Both types of structural anomalies are rectified by a series of addition, there is a group of patients with classic symptoms of XP enzymatic reactions referred to by the general term DNA repair but with normal excision repair. These are called XP variants (1-5). The repair reactions employed for correcting mismatches and (XP-V). Cells from XP-V patients are moderately sensitive to UV lesions are similar in principle. The incorrect or damaged base is light but excise UV photoproducts at a normal rate and are defec­ removed either as a base (base excision) or as an (oligo)nucleotide tive in a biochemically ill defined phenomenon called postreplica­ (nucleotide excision), the single-stranded gap resulting from the tion repair (10). excision reaction is filled in by a polymerase (repair synthesis), and Cockayne's syndrome patients suffer from growth failure, men­ the newly synthesized DNA is ligated. Hence, there are two basic tal and neurological abnormalities, cataracts, dental caries, and assays for measuring repair (6): the "incision/excision assay" and photosensitivity and related dermatoses. Mutations in two groups the "repair synthesis assay." of genes appear to cause Cockayne's syndrome. The CS-A and CS-B Excision Repair (ERCC-6) mutants exhibit classical CS symptoms without an in­ creased rate of skin cancer. Cells from these patients have near In base eXC1SlOn repair the mismatched or damaged base is normal UV sensitivity. A second group of patients manifest XP cleaved off the deoxyribose by a DNA glycosylase, and the resulting symptoms in addition to CS symptoms. Patients in this group have apurinic/apyrimidinic (AP)1 deoxyribose is released by sequential mutations in the XPB, XPD, or XPG genes. actions of an AP lyase which cleaves 3' and an AP endonuclease Trichothiodystrophy (TTD) patients have ichthyosis and brittle which cleaves 5' to the AP site. The one-nucleotide gap is filled in hair and suffer from photosensitivity, skeletal abnormalities, and and ligated (Fig. 1). mental retardation. The patients mayor may not have an in­ Nucleotide excision repair, conceptually, can be accomplished by creased rate of skin cancer. Mutations in three genes are associated two basic mechanisms. In one, a phosphodiester bond is hydrolyzed with TTD. In the XPITTD overlapping syndrome, the mutation is in 5' or 3' to the mismatch (lesion), and then the incorrect base is either XPB or XPD. In classical TTD (TTD-A), the mutation is removed by a 5' to 3' (or 3' to 5') exonuclease, which hydrolyzes presumably in one ofthe other subunits of TFIIH (1). DNA one nucleotide at a time starting at the nick and digesting In addition to the 9 genes identified by human diseases to be past the lesion. This is the repair mode employed by both Esche­ involved in excision repair, many rodent excision repair mutants richia coli and human general mismatch correction (repair) sys­ have been isolated and characterized in order to define the entire tems (1, 2). This endonuclease/exonuclease reaction pathway is not set of excision repair genes (11,12). The rodent mutants fall into 11 utilized for removing damaged bases from DNA. A possible expla­ complementation groups, and the majority of these correspond to nation for this is that most base adducts eliminated from DNA by human XP and CS complementation groups as indicated. In fact, excision repair inhibit exonucleases. One way to circumvent this some of the human XP genes were cloned by virtue of complement­ problem is to have an enzyme system that nicks the damaged ing rodent mutant cell lines and hence are also referred to as strand on both sides of the lesion at some distance removed from excision repair cross complementing (ERCC) genes. Ofthese genes, the lesion. This second mechanism, indeed, is the excision repair XPE and ERCC6 through ERCCll are not required for the basal mechanism found in all species investigated. As a matter of com­ excision reaction (13). mon practice, "excision repair" without further qualification means nucleotide excision repair of DNA damage, and hence it will be Structure and Function ofExcision Repair Proteins used as such in this review. Table I summarizes some of the properties of excision repair In excision repair, both procaryotes and eucaryotes hydrolyze proteins. Most of these proteins are in complexes in vivo, and hence the 3rd to 5th phosphodiester bond 3' to the lesion; on the 5' side the activity associated with a solitary protein in vitro mayor may the procaryotes hydrolyze the 8th (4) and the eucaryotes hydrolyze not be relevant to its function in excision repair. Human excision the 21st to 25th phosphodiester bond (7,8). Thus procaryotes excise nuclease has been reconstituted in a defined system by mixing six highly purified polypeptides or polypeptide complexes (13). * This minireview will be reprinted in the 1995 Minireview Compendium, XPA-This protein of 31 kDa has a zinc finger and is involved in which will be available in December, 1995. damage recognition (14). It also interacts with several other com­ :j:To whom correspondence should be addressed: Dept. of Biochemistry ponents of excision repair and hence may function as a nucleation and Biophysics, CB# 7260, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260. Tel.: 919-962-0115; Fax: 919-966-2852. factor for excinuclease. XPA interacts through its N-terminal do­ 1 The abbreviations used are: AP, apuriniclapyrimidinic; nt, nucleotide(s); main with the ERCCI-XPF heterodimer (6) to form a relatively XP, xeroderma pigmentosum; CS, Cockayne's syndrome; TTD, trichothiodys­ stable complex (15, 16); it also binds to TFIIH through its C­ trophy; ERCC, excision repair cross-complementing; RPA, replication pro­ tein A; HSSB, human single-stranded binding protein; TFIIH, transcription terminal domain (17). Finally, RPA (HSSB) binds to XPA and factor I1H; Pol, polymerase; PCNA, proliferating cell nuclear antigen. increases its specificity for damaged DNA (18). In addition to XPA, 15915 This is an Open Access article under the CC BY license. 15916 Minireview: Excision Repair in Mammalian Cells Base Excision Repair Nuc leoti de Excision Repair stoichiometry (13) an d bind to XPA through the N-terminal half of Damage and Mismatch Repair Damege Repair Mismatch Repair ERCC1 (15). Th e ERCC1-XPF complex is an endonuclease specific for single-stranded DNA.2 3 XPG-This protein has a single-stranded spec ific endonuclease .f'11111 811111 ' activity (34- 36). It also acts as a double-stranded specific exonu­ clease (34). It bin ds loosely to TFIIH (13) and to RPA (18) and is u ....,-/ ~ DNA Glycosylase (1) Endonucleas e (1) 3' or 5' nick apparently recruited by these compone nts to the excision nucl ease Excinuclea se (1) complex. 5' " 1 1 1 * " 1 " ~ Dual Incision Mechanism ofExcision Repair 5 ' ~ 3 ' Th e three forma l steps of excision repair are dam age recogniti on, AP Lyase (2) 1IIII Httr dual incision (excision), an d repair synthesis and ligation. I Damage Recognition- Excision repair was first identified by the 5' 1111 1'\11 11 111 ~ failure of UV-sensitive E, coli and human cells to remove thymine (2) 3E '-5x o n u 'c orl e a5'-3s e ' dimers from DNA. However, this repair syste m is not specific for JAP Endonuclease (3) nNMP. UV dam age as it excises all covalent DNA lesions test ed (37- 39). UMP 2 d A -' ~ With regard to substrate recognition an d preference, three inter­ 1 - 29 nl 2 ·300 SOOnl~ related questions mu st be addressed. Does the enzyme system 5' IIIII ~ IIIII ~ TITT-rrrr recogn ize only DNA with damaged bases, how does th e enzyme "know" which strand should be cut, and fina lly, what is the molec­ ~ (4 ) ular basis for recognition? ~ (3) Fir st , damaged bases are not the sole substrate for the enzyme . Human excinuclease excises mism atched bases and 1- 3-nt loops as 5' I I I I II ITIIT ~ well (38).