RETRACTED July 1, 2003

ICANCER RESEARCH57. 3784-3791, September I. 19971 Differential Involvement of the Human Mismatch Repair Proteins, hMLH1 and hMSH2, in Transcription-coupled Repair1

Steven A. Leadon2 and Anna V. Avrutskaya

Department of Radiation Oncology. University of North Carolina. Chapel Hill, North Carolina 27599-7512

ABSTRACT human cells, TCR of both UV and ionizing radiation-induced DNA damage requires the gene products defective in Cockayne syndrome Defects in DNA mismatch repair have been associated with both he. cells (1 1). We have recently shown that the TCR of oxidative damage reditary and sporadic forms of cancer. Recently, it has been shown that also requires a function of the XPG protein that is distinct from its human cell lines deficient in mismatch repair were also defective in the transcription-coupled repair (TCR) of UV-induced DNA damage. We endonuclease activity that is associated with NER (13). The XPG examined whether TCR of ionizing radiation-induced DNA damage also homologue in yeast, RAD2, also participates in TCR of oxidative requires the genes involved in DNA mismatch repair. Cells defective in the DNA damage; however, there is not an absolute requirement for hMSH2 gene were deficient in the removal of oxidative damage, including RAD2 in TCR, whereas there is a requirement for XPG in human cells thymine glycols, from the transcribed strand of an active gene. However, (12). The defect in TCR correlates with truncations of the XPG an hMLHJ mutant showed normal levels of TCR. By comparison, defects protein (16) that also lead to the clinical presentation of severe in either hMSH2 or hMLHJ resulted in reduced TCR of UV damage. Cockayne syndrome. Introducing chromosomes carrying either hMSH2 or hMLHJ into these Mutations in the genes required for DNA mismatch correction in cell lines restored their ability to carry out TCR. Deficiencies in either both E. co/i and human cells but not in S. cerevisiae have also been hMSH2 or hMLHJ did not result in decreased overall genomic levels of shown to selectively abolish TCR of UV damage (17â€”19).In both repair or lead to an increased sensitivity to either UV or ionizing radia tion. Our results provide the first evidence for a protein that is absolutely prokaryotes and eukaryotes, DNA mismatch repair plays a prominent required for the preferential removal ofUV-induced DNA damage but not role in the correction of errors made during DNA replication and oxidative DNA damage from the transcribed strand of an active human genetic recombination (20). In E. co/i, the methyl-directed mismatch gene. repair involves the products of the mutator gene mutS, mutL, mutH, and uvrD. In vitro, MutS is a DNA mismatch-binding protein, UvrD is DNA helicase II, and MutH is an endonuclease that incises at the INTRODUCTION transiently unmethylated DNA strand (20). No biochemical activity for MulL has been identified. Mutations in MutS and MutL but not NER3 is a mechanism that protects cells from the lethal, mutagenic, MutH result in a loss of strand-selective repair of UV damage, and/or carcinogenic consequences of otherwise persisting DNA dam implicating a role for these proteins in TCR (18). However, the age by bringing about the removal of several types of alterations in the addition of either MutS or MutL to an in vitro NER assay does not DNA (1). The removal of many types of DNA damage occurs in a July 1, 2003 affect the efficiency of lesion removal (21). TCR process whereby the damage is repaired more rapidly in Iran In eukaryotes, mismatch repair systems remain less well defined. scriptionally active DNA compared to the genome as a whole (2, 3). Genetic studies have demonstrated that the major DNA mismatch This rapid repair has been shown to be due to a faster repair of damage repair pathway in S. cerevisiae requires a bacterial MutS homolog, in the transcribed strand than in the nontranscribed strand of active MSH2 (22, 23), and two bacterial MutL homologs, MLH1 and PMS1 human genes. TCR of DNA damage seems to be a highly conserved (24, 25). Human homologues of the yeast mismatch repair genes pathway for excision repair, having been demonstrated in Escherichia ED hMSH2(26, 27), hMLHJ (28, 29), andhPMS2(30) have been iden coil (4, 5), Saccharomyces cerevisiae (6â€”8),and mammalian cells tified and shown to be mutated in patients and their kindreds with (9, 10). HNPCC. Tumors from patients with HNPCC have high mutation rates Although the original demonstration of TCR was made for DNA in microsatellite sequences (31), a hallmark of yeast with defective damage induced by UV, oxidative DNA damage, including thymine mismatch repair (32). Human tumor cells defective in either hMSH2, glycols, has also been found to be repaired in a transcription-coupled hMLHJ, or hPMS2 showed loss of TCR of UV damage, further process in both yeast and human cells (7, 1 1). Recently, we demon strengthening the link between TCR and mismatch repair (17). How strated that in a yeast rad1@@mutant (12) and in fibroblasts from ever, no defects in TCR of UV damage were found in yeast mutants xeroderma pigmentosum groups A and F (1 1, 13), all of which are for the MSH2, MLHJ, PMSJ, and MSH3 mismatch repair genes (19), defective in the NER pathway, oxidative DNA damage is preferen suggesting some functional differences between the yeast and human tially repaired in active genes. These results show that TCR can occur mismatch repair proteins. in the absence of NER and that repair of oxidative DNA damage In this study, we compared the repair of oxidative DNA damage in initiated by an N-glycosylase can be coupled to transcription. the transcriptionally active human metallothionein gene (Ml) in nor In E. coii, strand-selective repair is known to depend on the product mal and mismatch repair-deficient mutants. We showed that the of the mid gene product (14), which seems to displace RNA polym hMSH2 gene product, but not the hMLHJ gene product, was required erase stalled at a lesion, bind theRETRACT UvrA subunit of the excision for the preferential removal of oxidative DNA damage from the nuclease, and stimulate the repair of the transcribed strand (15). In transcribed strand of an active gene. However, both gene products were required for TCR of UV-induced DNA damage. In contrast to Received 4/I 8/97; accepted 7/3/97. The costs of publication of this article were defrayed in part by the payment of page previous results (17), deficiencies in either hMSH2 or hMLHJ did not charges. This article must therefore be hereby marked advertisement in accordance with lead to an increased sensitivity to either UV or ionizing radiation. 18 U.S.C. Section 1734 solely to indicate this fact. I Supported by USPHS Grant CA40453 from the National Cancer Institute. 2 To whom requests for reprints should be addressed. MATERIALS AND METHODS 3 The abbreviations used are: NER, nucleotide excision repair; TCR, transcription coupled repair; HNPCC, hereditary nonpolyposis colon cancer; BrdUrd, bromodeoxy Cell Culture Conditions and Treatment. Thecell lines usedin thisstudy, uridine; BrUra, bromouracil. SW480, HCT116 (clone A), HCT116 3-6, LoVo, HEC59, and HEC2-4C were 3784

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1997 American Association for Cancer Research. TCR IN MISMATCH REPAIR MUTANTS generously provided by the laboratory of Dr. Thomas A. Kunkel (National direction of transcription of the gene in vivo is from the BamHI site to the PvuII Institute of Environmental Health Sciences, Research Triangle Park, NC). Cell site. Strand-specific probes for the MT/A gene were generated using the two cultures were grown in DMEM/Ham's F12 medium containing 10% fetal phage RNA polymerase promoters, SP6 and 11, which are contained on bovine serum, 2 misiglutamine, and an antibiotic-antimycotic solution (Life pZMTIIA and oriented in opposing directions. Linearizing the plasmid with Technologies, Inc.). The HCT1 16 3-6 and HEC2-4C cell lines, which carry Hindu! and using T7 RNA polymerase and nbonucleotides produces a RNA human chromosomes 3 and 2, respectively, were grown in complete medium probe specific for the transcribed strand, whereas using BamHI and SP6 RNA supplemented with 400 @g/ml0418(Life Technologies, Inc.). Cultures that polymerase generates a RNA probe specific for the nontranscribed strand. were approximately 80% confluent were used in all experiments. For labeling Colony-forming Ability. For survival studies, irradiated cell cultures were of parental DNA, cells were grown for 3 days in medium containing 1 pCi/mI trypsinized and reseeded at appropriate densities into four to six 100-mm [3H]thymidine (Amersham) in the presence of 5 @tg/mlunlabeled thymidine. culture dishes/experimental condition. At the end of 7â€”10days,cells were The medium was removed, and the cells were grown for an additional 2 days stained with crystal violet, and colonies that contained >50 cells were counted in nonradioactive medium. Before irradiation, cells were incubated for I h in as survivors. Plating efficiency for control cultures ranged from 30â€”50%for medium containing 10 @MBrdUrdand 1 p.Mfluorodeoxyuridine. Cultures SW480, 9â€”12%for HCTI 16, 9â€”15%for HCTI 16 3-6, 7â€”12%forLoVo, were washed with PBS and irradiated with either a @Â°Coâ€˜y-sourceatdose rates 13â€”15%forHEC59, and 12â€”20%forHEC2-4C. of 1.6â€”1.8Gy/minor at 254 nm using a germicidal lamp at an incident dose rate of 0.65 J/m2/s. After irradiation, the cultures were either harvested imme diately or incubated for various lengths of time in medium containing 10 @M RESULTS BrdUrd and 1 @.LMfluorodeoxyuridine.For measurements of thymine glycol production and repair, cell cultures were exposed to 10 mr@iH202for 15 mm TCR of Ionizing Radiation Damage in Normal and Mismatch at 37Â°C.The cells were either harvested immediately, and the DNA was Repair-deficient Cell Lines. We examined the preferential repair isolated to determine the levels of thymine glycol in an ELISA using a of DNA damage in the MTIA gene using carcinoma cell lines that monoclonal antibody against thymine glycols (33), or they were incubated for are either mismatch repair competent or have alterations in the various lengths of time in medium. After incubation, the cultures were washed mismatch repair genes, hMSH2 and hMLHJ. Our approach in twice with PBS and harvested by lysis in 10 mM Tris, 10 mM EDTA, and 0.5% volves the physical separation of DNA regions containing BrdUrd SDS. substitution in repair patches from all other DNA using a mono Repair Analysis. Repair analysis of UV and ionizing radiation-induced clonal antibody against BrdUrd (35, 36). Cells were exposed to 10 DNA damage was carried out as described previously (11)using a monoclonal Gy of y-rays and either harvested immediately or allowed to repair antibody against BrUra. Briefly, purified DNA, digested with EcoRI, was in the presence of BrdUrd. Hybridization to the MT/A restriction centrifuged to equilibrium in a CsCl gradient to separate parental density DNA fragment from the bound and free fractions is shown in Fig. lA. (containing BrUra-substituted repair patches) from hybrid density DNA (syn Visual inspection of the autoradiogram shows that for most of the thesized by semiconservative replication; Ref. 34). Unreplicated, parental density DNA was then reacted with the monoclonal antibody against BrUra. cells examined, there is more of the transcribed strand of the MT/A The DNA bound by the antibody was separated by centrifugation. Aliquots of gene in the repaired (bound) fraction compared to the unrepaired the supernatant and pellet were assayed for radioactivity by liquid scintillation (free) fraction, whereas similar levels of the nontranscribed strand counting to determine the relative amount of DNA bound by the antibody. of the MT/A gene are found in both fractions for all of the cell lines Repair analysis of thymine glycols was carried out as described by Leadon examined.July These1, results2003 show preferential repair in the transcribed and Lawrence (7) using a monoclonal antibody that recognizes thymine strand of the MT/A gene compared to the nontranscribed strand. glycols in DNA (33). Heat-denawred DNA (50â€”100 @g)was incubated with Quantitation of the intensity of hybridization is presented in Fig. the antibody (1:1000 dilution) in PBS containing 0.1% BSA for 1 h at 37Â°C lB. Ionizing radiation-induced DNA damage is repaired more followed by an overnight incubation at 4Â°C.An equal volume of ice-cold rapidly in the transcribed strand of the MT/A gene in both the DNA saturated ammonium sulfate in PBS was added, and the mixture was incubated mismatch repair-proficient and -deficient cell lines (Fig. 18). How at 4Â°Cfor15 mm. The DNA bound by the antibody was collected as a pellet by centrifugation. Aliquots of the supematant and pellet were assayedED for ever, there is a decreased rate of repair in the two hMSH2 mutants, radioactivity by liquid scintillation counting to determine the relative amount LoVo and HEC59. Rapid rates of repair, similar to that found in the of DNA bound by the antibody. wild-type SW480 cells, were also found in HCT1 16, the hMLHI Equal amounts of DNA, based on the 3H prelabel, from the supernatant mutant, and in HCT1 16 3-6, which carries the wild-type copy of and pellet were electrophoresed on 0.7% neutral agarose gels. After dcc the hMLHI on chromosome 3. No differences in the repair of trophoresis, the DNA was transferred to a Gene-Screen Plus membrane damage on the nontranscribed strand of the MT/A gene or in (DuPont New England Nuclear). RNA probes were prepared as described overall genome repair between the cells were observed. Introduc by Leadon and Lawrence (10). After hybridization, the membranes were tion of wild-type hMSH2 on chromosome 2 corrected the defect in washed and exposed to KOdakXAR-5 X-ray film. The intensity of hybrid TCR in the HEC2-4C cells, as shown by a rapid repair of damage ization to the fragments of interest was measured by scanning densitometry on the transcribed strand of MT/A. Thus, it seems that the hMSH2 of the autoradiogram. The value for the density of each fragment was multiplied by the amount of DNA in the bound or free fractions to obtain gene but not the hMLHJ gene plays a role in the preferential repair the total amount of each gene in both fractions. The percentage of the MT/A of ionizing radiation damage in human genes. gene bound by the antibody was then calculated from the total amount of Strand-Selective Repair of UV Damage in Normal and Mis the gene in the bound fraction divided by the total amount of the gene in match Repair-deficient Cell Lines. The original observation im the bound plus free fractions. For the studies on the time course of repair plicating a role for mismatch repair proteins in the TCR of UV of thymine glycols, the percentage of theRETRACT genes containing thymine glycols damage pointed to a requirement for both the hMSH2 and hMLHJ immediately after treatment was set at 100%. gene products (17). However, we found that only the hMSH2 gene Probes. Results are presented for the MT/A gene, which is transcribed at product was required for the preferential repair of ionizing radia basal levels in all cell lines and tissues where it has been examined. The lion-induced DNA damage in an active gene. This discrepancy plasmid pZMTIIA, which contains most of the coding region of the human could be due to the different assays used. Therefore, we used our MTJJAgene, was constructed by subcloning a l75-bp BamHl-PvuIIcDNA assay for repair in specific DNA sequences to confirm the reported fragment isolated from the plasmid phMIH@(provided by M. Karin, Univer sity of California, San Diego, CA) into the BamHl-HincIl site of pGEM-3Z defect in the preferential repair of UV-induced damage in mis (Promega). Because of the extensive homology (at least 80%) of the multiple match repair mutant cells, which had previously been demonstrated members of this gene family to the coding region of MTIIA,this probe will using a method that measures pyrimidine dimers remaining in the hybridize to a 10-kb restriction fragment containing the MT/A gene. The DNA (17). The human cell lines were exposed to 10 JIm2 UV and 3785

0.5 1 Hours @ A. bf b1 I b f b f @@ ; @0TS @@ SW480 LoVo ,@â€¢â€¢ NTS

@ ai@ TS @@@ HCTII6 .-Â°.. @. HEC59 â€” NTS

@ HEC24C â€¢ . TS @ HCTII63-6 S., â€” â€” NTS

Fig. I. TCR in a restriction fragment containing the MT!Agene of mismatch repair-proficient and B. -deficient cells after exposure to ionizing radiation. Cells were exposed to 10Gy of 7-rays and allowed to repair in the presence of 10 @.sMBrCIUrd. Genomic DNA, digested with EcoRI, was reacted with an antibody to BrdUrd. DNAs from the bound (b) and free (1) fractionswere electrophoresedon 0.7% agarose gels and transferred to a Gene ScreenPlus membrane. A, representative autora C diograms from Southern analysis. B, kinetics of 0 to TCR in the MTJA gene. The percentage of total DNA (A) bound by the antibody was determined a) from the 3H prelabel. The percentage of the tran a scribed strand (TS; â€¢)and nontranscribed strand a, (NTS; 0) of the MT/A gene was determined by 0. densitometry of the autoradiogram. Points, the means of three to five experiments; bars, SE. July 1, 2003

20@ 20- LoVo ED HEC59 15 15- â€¢0 C

10-

I' @ V. â€¢ I 0 0.5 1 Post-TreatmentIncubation(Hr) RETRACT allowed to repair for various lengths of time. Hybridization of the (HCT1 16) and /iMSH2 (LoVo and HEC59) mutants were signifi probes for the transcribed and nontranscribed strands of the MT/A cantly reduced compared to that of SW480 cells. No differences gene to the restriction fragments from the bound and free fractions between the cell lines were observed in the rates of repair of UV is shown in Fig. 2A. The percentage of each strand of the MT/A damage in the nontranscribed strand of the MT/A gene or in the gene in the bound fraction is plotted in Fig. 2B. A more rapid repair genome overall. Introduction of the wild-type copy of the hMLHJ of damage on the transcribed strand of the MT/A gene is found in on chromosome 3 and hMSH2 on chromosome 2 restored the both DNA mismatch repair-proficient and -deficient cell lines ability of HCT1 16 3-6 and HEC2-4C cells, respectively, to carry compared to repair in the nontranscribed strand or the genome out TCR. Thus, cell lines with defects in the hMLHJ and hMSH2 overall (Fig. 2B). However, the rates of repair in both the hMLHJ genes show a reduction in but not a complete loss of their ability 3786

TCR tN MISMATCH REPAIR MUTANTS

3 6 3 6 Hours A b fb b I b f @@ TS SW480 LoVo @ â€œâ€¢Â°@- @.-# NTS

TS HCT116 HEC59 @ @. .$@ NTS

@@@ HCT1163-6 HEC2-4C . TS @@@ .@ NTS @ Â°@ *@.

Fig. 2. TCR in a restriction fragment contain B ing the MT/A gene of mismatch repair-proficient and -deficientcellsafterexposureto UV.Cells were exposed to 10J/m2 UV and allowed to repair in the presence of 10j.LMBrdUrd.Genomic DNA, digested with EcoRI, was reacted with an anti body to BrdUrd. DNAs from the bound (b) and â€¢0 free (J) fractions were electrophoresed on 0.7% C agarose gels and transferred to a GeneScreenPlus 0 membrane. A, representative autoradiograms from to Southern analysis. B, kinetics ofTCR in the MT/A C gene. The percentage of total DNA (A) bound by a) the antibodywasdeterminedfromthe 3Hprela a a) bel. The percentage of the transcribed strand (iS; 0@ â€¢)andnontranscribedstrand(N7S;0) of the MT/A gene was determined by densitometry of the autoradiogram. Points, the means of three to four experiments; bars, SE. July 1, 2003 0 3 6

LoVo

â€¢0 ED C 0 to C aa) a) 0.

0 3 6 0 Post-TreatmentIncubation(Hr)

to carry out TCR of UV-induced DNARETRACT damage. This defect can transcription-coupled process both in yeast (7) and, more recently, in then be restored by the introduction of their respective wild-type human cells ( 13). To determine whether the ionizing radiation TCR genes. defect in the hMSH2 mutants affected the removal of thymine glycols, Strand-Selective Removal of Thymine Glycols in Normal and we measured gene-specific removal of thymine glycols with an im Mismatch Repair-deficient Cell Lines. Oxidatively damaged bases munological approach that physically separates thymine glycol-con are the most abundant class of damage from ionizing radiation or mining DNA from all other DNA (7). other processes that generate reactive oxygen species, such as normal Cells were treated with 10 mr@iH2O, for 15 mm at 37Â°Cand cellular metabolism (37, 38). One of these is thymine glycol, a allowed to repair for 0.5 and 1 h. Using the monoclonal antibody to prevalent and lethal lesion that blocks transcription (39). We have thymine glycol in an ELISA (33), we found that these conditions previously demonstrated that thymine glycols can be repaired in a produce approximately I thymine glycol/lO kb (data not shown), 3787

TCR IN MISMATCH REPAIR MUTANTS

0 0.5 1 0 0.5 1 Hours A. b I b fb LoVo bfbfbf

SW48O@@ . â€”

@@ HEC59 @.. TS @@ HCT1 16

@@ HEC2 4C @Â°Â°@ TS HCT1 16 3-6 @@â€”@â€”â€”NTS

Fig. 3. Transcription-coupled removal of thy. B. 0) mine glycols from a restriction fragment contain C ing the MT/A gene of mismatch repair-proficient C and -deficient cells after exposure to H202. Cells E were exposed to 10mMH202 for 15 mm at 37Â°C. a, Purified DNA was digested with EcoRl and incu bated with an antibody against thymine glycols. Cl) and the extent of removal was determined as 8 described in the legend to Fig. 1. A. representative >â€¢ autoradiograms from Southern analysis. b, DNA a) bound by the antibody: f, DNA free of antibody. C B, kinetics for ICR of thymine glycols in the E MT/A gene. The percentage of total DNA (A) >â€¢ .C bound by the antibody was determined from the I- 3H prelabel. The percentage of the transcribed C strand (TS;â€¢)andnontranscribed strand (NTS:0) aa, of the MT/A gene was determined by densitome a) try of the autoradiogram. Points, the means of two to three experiments; bars, SE. g' i oo C E July 1, 2003 a) 80-

0@,60.

.@ 40- E â€˜C ED ); 20- C aa) a) 15 3- 0 0.5 1 0 Post-Treatment Incubation (Hr)

optimal for analysis of repair in the 10-kb EcoRI fragment on which strand of the MT/A gene. Thus, whereas there is only a reduction in MT/A is located. DNA from the H202-treated cells was incubated with the ability of hMSH2 mutant cells to perform TCR of ionizing radi the antibody to thymine glycol, and the antibody-bound DNA was ation damage, there is a complete loss of this repair subpathway in the separated from free DNA not containing thymine glycols. Hybridiza removal of thymine glycols. tion to the MT/A restriction fragment from the bound and free frac Cell Survival. We examined whether the defects in TCR in the tions is shown in Fig. 3A. Quantitation of the intensity of hybridiza DNA mismatch repair mutants used in our studies resulted in a tion is presented in Fig. 3B. AnalysisRETRACT of repair in the active MT/A gene hypersensitivity to killing by UV and ionizing radiation. The mis by hybridization of strand-specific probes revealed that thymine gly match repair-proficient SW480, HCT1 16 3-6, and HEC2-4C cells, the col is indeed removed much more rapidly from the transcribed strand hMLHJ mutant HCTI 16, and the two hMSH2 mutants, LoVo and in normal cells than from the nontranscribed strand or from total DNA HEC59, were irradiated with increasing doses of UV light, and their (Fig. 3B). Similarly, a rapid removal of thymine glycols was also colony-forming ability was assessed (Fig. 4A). In contrast to the found for the hMLHJ mutant cells (HCTI 16). In contrast, there is results reported by Mellon et a/. (17), no increased sensitivity in cell absolutely no preferential repair of thymine glycols in either LoVo or killing was observed in the mismatch repair mutants, although the HEC59, the two /MSH2 mutant cell lines. However, introduction of same cell lines were used in both studies. Similarly, exposure of the the wild-type /MSH2 gene on chromosome 2 restored the ability of mismatch repair mutants to increasing doses of ionizing radiation did HEC59 cells to rapidly remove thymine glycols from the transcribed not result in any hypersensitivity (Fig. 4B). Thus, the defects in the 3788

â€”a--LoVo

â€”Iâ€”HCT116 â€”*-. HEC59

@ A. â€”@â€”SW480 â€”0--HCT1163-6 -&-- HEC2-4C j

C,, C a) 0 a) 0@

Fig. 4. Sensitivity of mismatch repair-proficient and -deficient cells to UV and ionizing radiation. Survival curves for cells exposed to either UV (A) or â€˜y-rays(B) are shown. Irradiated cultures were reseeded into four to six 100-mm dishes. Points, the mean of four to six Dose(J/m2) experiments; bars, SE. B. @ 100' 100

@ 10' 10 C

a) a- July 1, 2003 I 1 1 I I 0 12345 Dose(Gy)

TCR of UV and ionizing radiation damage in the DNA mismatchED We demonstrated that there is a differential involvement of the repair mutants did not result in an increased sensitivity to killing by human mismatch repair genes in the preferential repair of ionizing these two agents. radiation damage. The hMLHJ mutant showed a fast initial rate of repair on the transcribed strand of the MT/A gene, similar to that found DISCUSSION with wild-type cells, indicating that the hMLHI gene product was not required for the preferential repair of ionizing radiation-induced dam In this study, we examined the role of two DNA mismatch repair age. However, the /ZMSH2 mutants showed decreased rates of repair proteins in TCR using six carcinoma cell lines that are either proficient or on the transcribed strand of the MT/A gene. Furthermore, when deficient in mismatch repair. SW480 has nonnal mismatch repair, removal of a specific type of oxidative DNA damage, i.e., thymine whereas HCF116, LoVo, and HEC59 are carcinoma lines that are de glycols, was examined, the hMSH2 mutants showed a complete lack fective in mismatch repair (40, 41). HCT1I6 has transversions in both of TCR. Because there was some residual strand-selective repair when alleles of hMU-J1 that result in the production of truncated polypeptides repair patches were measured, but not with the removal of thymine (29). The LoVo cells have deletions in both alleles of the !ZMSH2gene, glycols, these results suggest that there are types of ionizing radiation whereas FIEC59 has mutations in both alleles of this gene (42). A normal induced DNA damage that result in the production of repair patches copy of the hMLHJ gene on chromosome 3 has been transferred by microcell fusion into HCT1 16, resultingRETRACT in a correction of the mismatch but do not require the involvement of the !ZMSH2 gene product for repair deficiency and microsatellite instability in the HCT116 3-6 line their TCR. Clearly, removal of thymine glycols from the transcribed (43). Similarly, a normal copy of the /ZMSH2gene on chromosome 2 has strand of an active gene required the hMSH2 repair protein. Our been introduced into HEC59 and has been found to correct the defects in results provide the first evidence for a protein that is absolutely mismatch repair in the resulting HEC2-4C cell line.4 required for TCR of UV-induced DNA damage but not oxidative DNA damage.

4 A. Umar, M. Koi, J. I. Risinger, W. Glaab, K. R. Tindall, R. D. Kolodner, C. R. Whereas only the hMSH2 repair protein seemed to have roles in the Boland, J. C. Barrett, and T. A. Kunkel. Correction of hypermutability, N-methyl-N'- preferential repair of ionizing radiation-induced damage in active nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing genes, cells defective in either the hMLHJ or the hMSH2 genes had a chromosome 2 into human tumor cells with mutations in MSH2 and MSH6, submitted for publication. reduced ability to target repair of UV-induced damage to an active 3789

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1997 American Association for Cancer Research. TCR IN MISMATCH REPAIR MUTANTS gene. This suggests that although there is a role for these mismatch radiation or by H2O2-induced thymine glycols. Thus, a second refine repair proteins in TCR, there may not be an absolute requirement for ment is that if hMutLa, a heterodimer of hMLH1 and hPMS2, is these gene products in the preferential repair of UV damage. These required for the preferential repair of ionizing radiation-induced DNA results differ somewhat from those reported by Mellon et ai. (17) in damage in active genes, then hPMS2 may be able to fulfill that role in several respects. First, using an assay that measures the removal of the absence of a functional hMLHI . This would suggest that although cyclobutane pyrimidine dimers by loss of T4 endonuclease V-sensi the two MutL homologues seem to act as a heterodimer, inactivation tive sites, a total lack of TCR was found in the hMLHJ and !iMSH2 of each component could have different effects on TCR. Thus, if mutants compared to our findings of reduced levels (17). Because the hPMS2 alone could facilitate strand-selective repair of oxidative DNA same cell strains were used in both studies, this apparent discrepancy damage in the absence of a functional hMLHI protein, it would be could be due to differences in how measurements of repair in a predicted that both hMLHI and hPMS2 proteins would need to be specific gene were made. In our assay, an antibody that recognizes inactivated to detect a defect in TCR of oxidative damage. This latter BrdUrd is used to detect the repair patch itself, regardless of the type interpretation is supported by our observations in yeast that there is a of UV damage that is being repaired. Thus, the differences could defect in TCR of thymine glycols in MSH2 mutants and in MLHI/ reflect a requirement of the mismatch repair proteins in the repair of PMSI double mutants, but not in single MU/i and PMSJ mutants.5 some, but not all, UV-induced photoproducts, such as the cyclobutane In summary, we have shown that only the hMSH2 gene product was pyrimidine dimers, but not the (6â€”4)dipyrimidines. required for the TCR of oxidative DNA damage, whereas both the Inactivation of the hMSH2 and hMLHI genes, which results in /ZMSH2 and hMLHJ gene products were required for the TCR of decreased TCR, apparently does not lead to an increased sensitivity of UV-induced DNA damage. However, mutants with either of these the mutant cells to UV or y-rays. These results, at least with respect mismatch mutations did not show decreased overall levels of DNA to UV, are in contrast to those of Mellon et ai. (17), in which a repair or an increased sensitivity to killing by UV or ionizing radia significant increase in UV sensitivity of the mismatch repair mutants tion. These results provide the first evidence for an absolute differ was reported. This suggests that an increased sensitivity to a DNA ential involvement of a protein in TCR of ionizing radiation- and damaging agent may not be a reproducible end point for characteriz UV-induced DNA damage and implicate unrepaired oxidative DNA ing mismatch repair mutants. damage, such as that produced during normal cellular metabolism, as DNA mismatch repair could play at least two roles in TCR: a contributing factor in the etiology of certain sporadic and hereditary (a) Mismatch repair proteinsmight ensurethe fidelity of repair of cancers, e.g., HNPCC. damage in an active gene by removing errors introduced during DNA repair synthesis. The presence of factors involved in either transcrip REFERENCES tion or repair could serve as strand discrimination signals for mis match repair. Indeed, it has recently been shown that proliferating cell I. Friedberg. E. C.. Walker, G. C.. and Siede, W. DNA Repair and Mutagenesis. Washington, DC: American Society for Microbiology Press, 1995. nuclear antigen, which is required for NER in vitro (44, 45), also 2. Hanawalt, P. C. Transcription-coupled repair and human disease. Science interacts with both yeast MLH1 and MSH2 (46). However, this model (Washington DC), 266: 1957â€”1958,1994. 3. Friedberg, E. C. Relationships between DNA repair and transcription. Annu. Rev. would not account for a reduction in lesion removal in mismatch JulyBiochem., 65:1, 15â€”42,1996. 2003 repair mutants observed for UV-induced cyclobutane pyrimidine 4. Mellon, I., and Hanawalt, P. C. Induction of the Escherichia coli lactose operon dimers and H2O2-induced thymine glycols. selectively increases repair of its transcribed DNA strand. Nature (Lend.), 342: 95â€”98,1989. (b)Analternativebutnotmutuallyexclusiveroleformismatch5. Kunala, S., and Brash, D. E. 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Retraction

Methodologic errors in assays of transcription-coupled DNA repair reported in an article published in the September 1, 1997, issue of Cancer Research (Leadon, S. A., and Avrutskaya, A. V. Differential involvement of the human mismatch repair proteins, hMLH1 and hMSH2, in transcription-coupled repair. Cancer Res., 57: 3784–3791, 1997) have called into question the validity of some of the findings in the study. The authors have therefore requested that the article be retracted from the literature.

3846 Differential Involvement of the Human Mismatch Repair Proteins, hMLH1 and hMSH2, in Transcription-coupled Repair

Steven A. Leadon and Anna V. Avrutskaya

Cancer Res 1997;57:3784-3791.

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