Base Excision Repair of U:G Mismatches at a Mutational Hotspot

(CANCER RESEARCH 55, 3742-3746, September1, 1995]

Advances in Brief

Base Excision Repair of U:G Mismatches at a Mutational Hotspot in the p53 Gene Is More Efficient Than Base Excision Repair of T:G Mismatches in Extracts of Human Colon Tumors1

Christoph Schmutte,2 Allen S. Yang, Robert W. Beart, and Peter A. Jones

Department of Biochentistn- and Molecular Biolog\, USC/Norris Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles, California 90033-0800

Abstract mutation is the methylation process itself, which we have shown to be the case for bacterial methyltransferases (IO).4 In an impressive study, Approximately 50% of mutations that inactivate the p53 tumor sup Laird et al. (11) have recently provided new evidence supporting this pressor gene in the germline and in colon tumors are C to T transitions at enzymatic pathway for cytosine deamination. methylation sites (CpG sites). These mutations are believed to be caused by an endogenous mechanism and spontaneous deamination of 5-methyl- On the other hand, spontaneous hydrolytic deamination of C and cytosine to T is likely to contribute significantly to this high mutation rate. 5mC are generally accepted to be a major source of transition muta The resulting T:G mismatches created by this process have been hypoth tions (12-15). Deamination of C leads to U, creating a U:G mismatch, esized to be less efficiently repaired than U:G mismatches formed by whereas 5mC deaminates to T, forming a T:G mismatch (Fig. 1). Both deamination of C. We have, therefore, performed the first study to U and T pair with A during replication, resulting in a C to T mutation directly compare rates of T:G versus U:G base excision repair at identical in the next cell generation if unrepaired (Fig. 1). The rates of spon sites observed to be mutated in the p53 gene using extracts of human taneous deamination of C and 5mC in double-stranded DNA have normal colon mucosa and colon carcinoma tissue. Mismatched U was been measured under physiological conditions to be about excised up to 6000-fold more efficiently than T, suggesting that differences 2.6 X 1CT13 s~' for C and 6 X 10~'3 s~' for 5mC (16, 17). in repair efficiencies are the major source of C to T transition mutations Therefore, approximately 500 5mC to T and about 200 C to T at CpG sites in human tissues. The data also suggests that T:G mismatches deaminations per 10'" bp would be expected per day. Both numbers are repaired by additional mechanisms in human cells. are more than sufficient to explain all of the transition mutations seen Introduction in human tumors if the premutagenic mismatches are not repaired (Fig. 1). Nevertheless, far less mutations in total are observed, most of Mutations in key genes, including activating mutations in proto- these mismatches must get repaired, and the efficient repair of U:G oncogenes or inactivating mutations in tumor suppressor genes, are and T:G mismatches is, therefore, crucial for normal cellular function. thought to play a major role in the formation and progression of However, deamination rates alone cannot explain the 42-fold higher human cancers (1). Examination of the spectrum of mutations in rate of cytosine deamination seen at CpG sites as compared to non- tumor suppressor genes, in particular p53, the most frequently mu CpG sites. tated gene in human tumors, has provided insight into the nature and It has been proposed (18) that U:G mismatches are repaired more cause of these mutations (2, 3). Human tumors of different origins often show tissue-specific patterns of mutation types in the p53 gene. readily than T:G mismatches since T, unlike U, is a normal compo nent of DNA. Furthermore T:G mismatches cause little distortions of Transversions from G to T, which are prevalent in lung tumors, are the DNA helix (19) and are thus more difficult to be recognized by thought to be caused by exogenous carcinogens present in tobacco cellular repair enzymes. This could explain why transitions are found smoke [reviewed in Greenblatt et al. (4)]. In contrast, colon carcino to a much higher extent at methylated CpG sites compared to Cs, mas contain a high percentage of C to T transitions in p53, and 75% which are 5' to other bases. of these transitions (i.e.. 47% of all mutations) occur at the CpG The first and rate-limiting step of base excision repair is carried out dinucleotide in this tissue type, although only 39 of the total of 512 by DNA glycosylases that excise the damaged or mismatched base to cytosines in the p53 cDNA are within CpG sites. In addition, about 50% of all germline mutations in p53 (Li-Fraumeni Syndrome) com leave abasic sites (AP). AP sites are subsequently cleaved by an AP-endonuclease and deoxyphosphodiesterase (14, 20) to create a prise C to T or G to A transitions at CpG, consistent with an single nucleotide gap, which is then filled in by a DNA polymerase, endogenous mechanism for mutagenesis. Five of the six mutational hotspots in the p53 gene are CpG sites, and sealed by DNA ligase (21). Mammalian cells contain a series of and all of these are methylated to 5mC3 in every tissue examined DNA glycosylases, each of which removes a specific lesion. UDGs, (5-8). Cytosine methylation within a CpG dinucleotide is, therefore, which initiate U:G base excision repair, are ubiquitous and well characterized (22-24). UDGs hydrolyze the /V-glycosidic bond linking likely to be causally involved in creating the increased mutation rate at these sites (9). One possible mechanism to explain this type of the base to the deoxyribose sugar (20, 25). Base excision repair of T:G mismatches within CpG sites is initialized in a similar mechanism by a TDG, which has been identified in extracts from HeLa cells by Received 6/5/95; accepted 7/24/95. The cosls of publication of this article were defrayed in part by the payment of page Wiebauer and Jiricny (26, 27) and Neddermann and Jiricny (28). This charges. This article must therefore be hereby marked advertisement in accordance with enzyme has been proposed to counteract loss of CpG sites in a similar 18 U.S.C. Section 1734 solely to indicate this fact. ' This work is supported by Grant R35 CA49758 from the National Cancer Institute. way to the bacterial vsr repair system for dem methylation sites 2 To whom requests for reprints should be addressed, at Kenneth Morris Jr. Compre (CCWGG; Ref. 29). hensive Cancer Center, University of Southern California, P.O. Box 33800. 1441 Eastlake Avenue, Los Angeles, CA 90033-0800. 1The abbreviations used are: 5mC. 5-methylcytosine; UDG, uracil-DNA glycosylase; 4 J-C. Shen. J-M. Zingg. A. S. Yang, C. Schmutte, and P. A. Jones. A mutant DNA TDG, thymine-DNA glycosylase; AP, abasic site; vsr, very short patch repair. (cytosine-5)-methyltransferase functions as a mutator enzyme, submitted for publication. 3742

Normal Premutagenic Base Pair Lesion Mutation

Repair: i. Base Excision Repair (TOC) 2. Nucleotide Excision Repair (?) 3. Long Patch Repair (hMutHLS) X CpGsite: 5mC:G T:G

Fig. 1. Proposed mechanism for Ihc formation of C to T transition mutations and its repair. [H20] T:A

non-CpG site: V Repair: 1. Base Excision Repair (UDG) 2. Nucleotide Excision Repair (?) 3. Long Patch Repair (hMutHLS) (?)

We report in this paper the first quantitative comparison of base normal mucosa. Oligomers of identical sequence but containing either excision repair rates of U:G versus T:G mismatches in the same a U:G or T:G mismatch at position 13 were 5'-end labeled on the sequence context by extracts of human colon tissue. Our results using strand containing U or T, respectively, which were mismatched with the identical sequence for the codon 248 mutational hotspot in the p53 G. Incubation with cell extracts for varying times resulted in excision gene demonstrate U-excision is, by several orders of magnitude, more of the mismatched base, and the resulting abasic (AP) site was efficient compared to excision of T in human colon tissue extracts. efficiently cleaved by AP endonucleases present in the extract. Com They support a model that mutational hotspots at CpG sites in human plete cleavage had been confirmed by treating the oligomers with colon tissue are mainly caused by differences in repair efficiencies of alkali. The shortened labeled strands could then be easily detected these premutagenic lesions. They also suggests that T:G mismatches after electrophoresis on a denaturing gel and autoradiography. Fig. 2 might get repaired by other repair mechanisms in addition to base shows results obtained for UDG excision by HeLa cell extracts or excision repair. extracts of normal appearing colonie epithelium and adjacent tumor. No cleavage of the labeled 24-mer occurred with oligomers contain Materials and Methods ing C or U at the target site, incubated for l h without extract. The extracts each behaved similarly in inducing a concentration-dependent Colon Specimen and Cell Lines. Human colon cancer tissue and sur rounding normal mucosa were collected from cancer patients undergoing excision of the U, resulting in the formation of a 13-mer. None of the coloectomy. Tissue was frozen in liquid nitrogen or in ethanol/dry ice within extracts induced cleavage of the labeled oligomer containing a C at 30 min after coloectomy and kepi at â€”70Â°Cuntil use. The tumors analyzed the target site. The correct identity of the 13-mer was confirmed by were of various grades and stages, and all had a diameter of more than 0.5 inch. incubating the oligomer with purified UDG (Boehringer Mannheim), Cell lines were obtained from American Type Culture Collection (Rockville, which resulted in a signal at a similar position on the gel after alkali MD) and used between passage numbers 50 and 120. lysis (data not shown). In addition, treatment of the C:G oligomer with Determination of UDG Activity and TDG Activity. Glycosylase activi the restriction enzyme Msp\ resulted in the formation of a 12-mer that ties were measured using a slightly modified method developed by Wiebauer and Jiricny (26) and improved by Griffin and Karran (30). Oligomers (5'- ran slightly further on the gel (data not shown). These results, there GGCTATCGTGGCXGGCCACGACGG-3': X = C, U, or T, respectively) fore, showed that all three cell types examined could rapidly remove were synthesized, gel purified, and 5'-end labeled using [-y-'-PjATP (Amer- U from the U:G mismatch in a concentration-dependent manner. sham) and T4 polynucleotide kinase (Boehringer Mannheim). U:G or T:G Base excision repair of T:G mismatches in the same sequence mismatches were created by annealing with unlabeled 5'-CCGTC- context was measured next (Fig. 3). Treatment of the U:G oligomer GTGGC5mCGGCCACGATAGCC-3'. Total cell extracts from human colon with HeLa extract lead to a complete cleavage of the oligomer after specimens were prepared by homogenization in extraction buffer containing 20 incubation for 16 h. However, only about one-half of the oligomer HIM HEPES (pH 7.5), 2 itiM EDTA, 10 HIM EGTA, 2 mM DTT, 0.5 mM containing a T:G mismatch at the same position was cleaved after this phenylmethylsulfonyl fluoride, and 10 /xg/ml leupeptin. Double-stranded oli- incubation time. Further dilution of the extract resulted in a concen gomcrs (0.5 pmol) were incubated with extracts (4 Â¡j.gprotcin/fxl) in buffer tration-dependent decrease in the amount of cleavage product. No T:G containing 50 mM 1,4-piperazineethanesulfonic acid (pH 6.7), 10 JU.MZnCU,0.5 mM EDTA, and 1 mM DTT al 30Â°Cin a final volume of 25 /il. The reaction was mismatch excision was detectable after l h of incubation, whereas stopped by adding formamide stop solution. Oligomers were separated by 12% U:G repair has been completed using the same amount of template denaturing PAGE and quantitated by scanning the radioactive signals using a (data not shown). We subsequently used 2 /j,l of extract of colon tissue radio-analytic imaging system (AMB1S, Inc., San Diego, CA). Complete cleavage to measure T:G and U:G mismatch excision in a series of colon of the abasic site created by the glycosylase reaction was verified by treatment of carcinoma and normal mucosa (Fig. 4). As seen with HeLa cell extract samples at 90Â°Cin basic solution. Protein concentrations were measured using (Fig. 4, Â¿Ã/rtfÃ5and 6), excision of U was completed after 16 h, Bio-Rad protein assay (Bio-Rad). whereas excision of T was about 50% complete for the same incuba Results tion time. All of the tissue samples studied were capable of both U:G and T:G excision; however, both activities were decreased relative to Repair efficiencies of U:G and T:G mismatches were compared HeLa cells. All patient samples showed a significantly greater ability using extracts of HeLa cells, colon carcinoma tissue, and adjacent to excise U compared to T. AP sites cleaved by excision 5' to the 3743

normal colon HeLa colon mucosa carcinoma oligomer- C U C U u u in in in in extract^!) w in c\i i- o ini evi -r- p 10 C\l i- O Ã³ ci Ã¶ o ci o Ã³ Ã³ Ã¶ Ã³ Ã³ Ã³

Fig. 2. Concentration-dependent repair of U:G - 24mer mismatches. Oligomers containing a C:G and U:G hp, respectively, were incubated with extracts from human colon tissue or HeLa cell extract. After 1 h, oligomers were subjected to alkali lysis and subse quently separated by denaturing 12% PAGE. *, base paired with G.

cleaved ^^^^Â»Â»4^^fe ^^ _ ^k^fe^^^few^p^w & oligomers

extract 0.5 0.25 the same range (data not shown). Oligomers containing mismatches in oligomer- CUT UT UT UT UTUT the exact sequence context of p53 codon 248, which is a mutational hotspot for C to T transitions in human colon carcinomas (CAT- 24mer â€” GGGCGGCATGAACXGGAGGCCCATCCTCAC; X = C, U, or T), were also used, and essentially the same results were obtained (data 13mer â€” not shown). In addition, we measured UDG and TDG activities in total cell Fig. 3. Concentration-dependent repair of U:G and T:G mismatches. Oligomers con extracts of four colon carcinoma cell lines (SW480, SW837, HCT15, taining C:G, U:G, and T:G hp, respectively, were incubated with HeLa cell extract for 16 h at 30Â°Cand subsequently separated by denaturing PAGE. *. base paired with G. and HCT116). Interestingly, extracts from SW480 showed very little TDG activity, whereas UDG activity was comparable to HeLa cell extracts (Fig. 5C). We found no significant difference in base excision mismatch and subsequently by incision 3', removing the deoxyribose, activity for T:G and U:G mismatches, respectively, among total cell resulted in the appearance of 12-mers seen as a second product on the extracts of SW837, HCT15, HCT116, and HeLa cells. gel. The same pattern has been shown previously for HeLa cell In summary, base excision repair of T:G mismatches was about extracts by Wiebauer and Jiricny (26) and Griffin and Karran (30), 600- to 6000-fold slower than excision of U from U:G mismatches in respectively. extracts from colon tissues and cell lines. These results support the Mismatch excision data from seven patients are summarized in Fig. hypothesis that differences in repair efficiencies of U:G versus T:G 5. Amounts of cleaved oligomer were determined by scanning the mismatches are the main reason that CpG sites are mutational hotspots radioactive bands on the polyacrylamide gels. Activities measured in human tumor suppressor genes. were approximately 300 fmol//j.g protein/min for UDG and 0.18 fmol/p,g protein/min for TDG in colon tissue extracts. UDG activity Discussion was, therefore, about 600 to 6000-fold more active than TDG in these tissues. No significant differences were seen between normal and It is generally accepted that the high percentage of C to T or G to cancerous tissue (Fig. 5, A and B). A mucosal extract from a patient A transition mutations at CpG sites seen in human tumor suppressor without cancer showed UDG and TDG efficiencies, respectively, in genes in several human cancer types are caused by an endogenous

B - HeLa extract N N N CUTCUTCUTCUTCUTCUTCUTCUT oligomer"

24mer Fig. 4. Comparison of U:G and T:G mismatch excision in extracts of human colon tissue and HeLa cells. Oligomers were incubated with extracts (2 fil in a total volume of 25 /Â¿I)for 16 h and separated on a denaturing polyacrylamide gel as described in Fig. 2. *, patient (N, normal; T. tumor tissue). **, base paired with G.

55 â€¢ ?- cleaved Oligomers

3744

900- UDG activity is ubiquitous in human tissue. It has been shown that U is efficiently excised from SV40 DNA by simian UDG (34). Although it seems likely that the predominant route to transition mutations at CpG sites is through to T:G mismatch, we have shown recently that U:G mismatches can be shielded from UDG by methyltransferase, forming a tight binding complex with methylation sites containing U:G mismatches (35). There is still, therefore, the possibility that some premutagenic lesions may be masked from efficient repair enzymes by proteins bound to DNA. T:G mismatch repair in eukaryotic cells is less well understood. Unlike U, T is a normal DNA base, which makes the strand bias in the repair process a crucial question since excision of G would complete a C to T mutation. Brown and Jiricny (36) demonstrated that T:G mismatches are repaired with more than 99% efficiency in green B UDG 400- monkey (CV1) cells, with 90% in favor of C:G. Griffin and Karran D TDG (30) have shown that the glycosylase, which had been discovered in 300- HeLa cell extracts (26), excises T from T:G mismatches within CpG 200- sites. The enzyme shows highest activity when the opposite C is 100- methylated. Therefore, this enzyme counteracts loss of CpG sites by spontaneous deamination in the human genome. Interestingly, we found no consistent differences between excision 0,2- in normal and tumor tissue (Fig. 5). This suggests that inactivation of this repair enzyme does not correlate with the development of colon cancer in these patients, as has been shown for the human mutHLS repair system (hMSH2 and hMLHl), which is inactivated in a high uuupercentage of hereditary nonpolyposis colon carcinoma cases (37- 40). In bacteria, this repair system is methyl-directed and repairs 700- misincorporated bases directly at the replication fork before the nas 600- cent DNA strand becomes methylated (41, 42). It is not clear if this repair system also repairs T:G mismatches formed by deamination of 500- 5mC in addition to T, which has been inserted opposite G during i 400 - replication (Fig. 1). This premutagenic lesion has to be repaired before 300- the cell enters S-phase. Jiricny et al. (43) have discovered a Mr 200,000 protein that binds selectively to T:G mismatches, although 200- the binding is not CpG specific. Since the sequence is homologous to the bacterial mutS gene (hMSH2; Ref. 44), this protein most likely interacts with human homologues of MutL and MutH in the methyl- directed pathway.5 On the other hand, it has been shown that nicks in DNA can function as a strand-directing signal (45). Thomas et al. (46) HeLa SW480 SW837 HCT15 HCT116 found that cellular HeLa extracts can repair T:G mismatches if the Fig. 5. Summary of specific base excision from U:G and T:G mismatches, respectively, T-containing strand is nicked. As mentioned before, bacterial enzyme by extracts from normal human colon mucosa (A), colon carcinoma tissue (ÃŸ),and vsr initiates repair of T:G mismatches within a methylation site (dem; various cell lines (C). CCWGG) by nicking the T-containing strand and, in that way, coun teracts loss of these sites in bacteria (47, 48). E. co/i-defective in vsr shows an elevated spontaneous mutation rate (49, 50). A human mechanism (4, 5, 15). Since CpGs are methylation sites, and all homologue of the vsr enzyme has not been characterized yet; how mutational hotspots in the p53 tumor suppressor gene are methylated ever, a nicking activity for different mismatches has been described in to 5mC, the hydrolytic deamination of this base forming a T:G HeLa cell nuclear extracts by Yeh et al. (51). The rate of T excision mismatch may contribute significantly to the formation of these we measured in nuclear HeLa cell extracts was of the same magnitude mutations (12, 18). as that described by Wiebauer and Jiricny (27) and Griffin and Karran Stability of C and 5mC in double-stranded DNA has been deter (30). It is surprising that this repair is so slow. It is unlikely that other mined in vitro under physiological conditions, and the spontaneous proteins are essential in the repair process performed by TDG since deamination rate of 5mC is 2.2-fold higher than the rate of C (16, 17). the enzyme is active in a highly purified form (28). The enzyme is also As pointed out by Lindahl (14), this relatively small difference is by able to excise mismatched U, which might be its main function (52). itself unremarkable, but this effect is thought to be amplified by As mentioned earlier, transition mutations at 5mC occur 40 times differences in repair rates of U:G and T:G mismatches, which are more frequently than at C in the p53 gene, and 5mC deaminates about formed by the deamination process. In fact, this has been shown for 2.6 times faster than C. On the other hand, T is excised from T:G repair of these mismatches in Escherichia coli (31). mismatches three orders of magnitude slower than U from U:G U:G mismatches seem to be repaired predominantly by base exci mismatches. It is, therefore, likely that other mechanisms contribute to sion repair in bacterial and mammalian cells, and three UDGs have the repair of T:G mismatches at CpG sites (Fig. 1). Further studies been characterized in human cells (22-24, 32). In addition, other glycosylases, which excise bases with very similar structures to U 5 This has been shown lo be the case (Drummond el at.. Science (Washington, DC), such as hydroxymethyluracil-DNA glycosylase, also excise U (33). 268. 1909-1912, 1995). 3745

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. BASE EXCISION REPAIR OF U:O MISMATCHES have to be done to clarify this point. Since the assay system we used sylase: structural basis for specificity and catalysis. Cell. 80: 869-878, 1995. 26. Wiebauer, K-, and Jiricny. J. In vitro correction of GT mispairs to GC pairs in nuclear to measure U:G and T:G mismatch repair only scores base excision extracts from human cells. Nature (Lond.). 339: 234-236, 1989. repair, other repair systems present in the mammalian cell may change 27. Wiebauer. K., and Jiricny. J. Mismatch-specific thymine DNA glycosylase and DNA the repair efficiencies of T:G versus U:G presented here. polymerase ÃŸmediate the correction of (Â¡Tmispairs in nuclear extracts from human cells. Proc. Nati. Acad. Sci. USA. 87: 5842-5845. 1990. In summary, these data support the model that differences in repair 28. Neddermann. P.. and Jiricny. J. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. Base Excision Repair of U:G Mismatches at a Mutational Hotspot in the p53 Gene Is More Efficient Than Base Excision Repair of T:G Mismatches in Extracts of Human Colon Tumors

Christoph Schmutte, Allen S. Yang, Robert W. Beart, et al.

Cancer Res 1995;55:3742-3746.

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