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MSH2-Deficient Human Cells Exhibit a Defect in the Accurate Termination of Homology-Directed Repair of DNA Double-Strand Breaks1

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MSH2-Deficient Human Cells Exhibit a Defect in the Accurate Termination of Homology-Directed Repair of DNA Double-Strand Breaks1 [CANCER RESEARCH 63, 3334–3339, June 15, 2003] MSH2-deficient Human Cells Exhibit a Defect in the Accurate Termination of Homology-directed Repair of DNA Double-Strand Breaks1 Jose´e-France Villemure, Christine Abaji, Isabelle Cousineau, and Abdellah Belmaaza2 Department of Biochemistry, Universite´ de Montre´al, Succ. Centre-Ville, Montre´al, Que´bec, Canada H3C 3J7 [J-F. V.], and Department of Biochemistry [C. A.], Molecular Biology Program [I. C.], and Department of Microbiology and Immunology [A. B.], Universite´ de Montre´al, Centre de Recherche du CHUM, Hoˆpital Notre-Dame, Institut du Cancer de Montre´al, Montre´al, Que´bec, Canada H2L 4M1 ABSTRACT of small IDLs as well as larger IDLs, containing up to 12 nucleotides (9, 10). The relatively low number of hMSH6 and hMSH3 mutations Mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 in HNPCC kindreds has been attributed to their imparted redundancy have been associated with hereditary nonpolyposis colorectal cancer. Tu- in substrate specificity. hMLH1 can form a heterodimer with hPMS2, mor cell lines that are deficient in MMR exhibit a high mutation rate, a defect in the response to certain types of DNA damage and in transcrip- hPMS1, or hMLH3 (11). The three types of hMutL complexes are tion-coupled repair, as well as an increase in the rate of gene amplifica- presumably functionally redundant. hMutS plays a central role in tion. We show here that hMSH2-deficient tumor cell lines lost most of initiating mispair recognition and binding, and hMutL acts as a their ability to accurately repair plasmid DNA double-strand breaks molecular matchmaker between hMutS and downstream proteins to (DSBs) by homologous recombination, compared with MMR-proficient or complete the repair process (1–5). Therefore, both hMSH2 and hMLH1-deficient tumor cell lines. In all of these cell lines, DSB repair hMLH1 are critically important to MMR activity, as reflected by their occurred almost exclusively by nonreciprocal homologous recombination: predominant alteration in HNPCC patients, as well as in most sporadic gene conversion (GC). However, there were two types of GC products: tumors with microsatellite instability. precise and rearranged. The rearranged products contained deletions or The majority of cells deficient in MMR exhibit a mutator pheno- insertions of sequences and represented GC intermediates trapped at type characterized by a 100-1000-fold increment in the rate of spon- various stages and shunted to nonhomologous end joining. In MMR- taneous mutation at miscrosatellites and coding sequences (12–15). proficient or MLH1-deficient cells, >50% of GC products were of the precise type, whereas in two MSH2-deficient backgrounds, this propor- However, studies on mice with MMR gene knockout indicate that tion decreased to 8%, whereas that of rearranged GC products increased mutations of MMR genes other than hMSH2 and hMLH1 may induce by 2-fold. These results seem to predict a novel way by which MSH2- a mutator phenotype but not necessarily an increase in colon tumors, deficiency could promote mutation: deletion or insertion mutations implying that a mutator phenotype alone may not be sufficient for associated with DSB repair, which may also contribute to cancer predis- intestinal tumor formation (16). In addition to editing replication, position. MMR proteins have also been implicated in the editing of recombi- nation between divergent sequences, in transcription-coupled repair of INTRODUCTION DNA damage, and are also thought to function as lesion sensors for certain types of DNA damage that kill by triggering apoptosis (1, 3–5, MMR3 is a highly conserved repair system that corrects mis- 17). Moreover, MMR proteins inhibit gene amplifications (18, 19) and matches arising during DNA replication and safeguards genomic have been associated with the fidelity of DNA DSB repair, although integrity (1–5). Defective MMR is strongly associated with HNPCC, the underlying mechanisms remain unknown (19–23). an autosomal dominant inherited disease characterized by early onset DNA DSBs are common lesions that occur in all of the cells, and colon tumors, as well as cancers of the endometrium, stomach, upper DSB repair is a fundamental mechanism of genome protection. There urinary tract, small intestine, and ovary (6, 7). MMR consists of at are two major DSB repair pathways in mammalian cells: homologous least seven proteins, including four bacterial MutL homologues, recombination and NHEJ (24). Defects in either pathway have been hMLH1, hMLH3, hPMS1, hPMS2, and three MutS homologues, linked with genome instability and cancer (25–27). In mammalian hMSH2, hMSH3, and hMSH6. Defects in hMSH2 and hMLH1 appear cells, DSB repair by homologous recombination occurs almost exclu- to be the cause of most HNPCCs (8). sively by GC: a nonreciprocal transfer of genetic information from an MMR proteins function as heterodimeric complexes. hMSH2 can intact homologous duplex to a broken duplex (28–30). In this path- form a heterodimer with either hMSH3 or hMSH6. hMSH2 is a way (31), DSBs are first processed to yield 3Ј-ended single-strand uniformly essential component of all of the heterodimers, whereas tails (Fig. 1a) that invade a homologous donor duplex (Fig. 1b). DNA hMSH3 and hMSH6 modify substrates specificity. hMSH2-hMSH6 synthesis is then primed from the 3Ј end of the invading strand, which heterodimer recognizes single bp mismatches and small IDLs, results in the copying of donor information (Fig. 1c). DSB repair may whereas hMSH2-hMSH3 heterodimer recognizes an overlapping set be completed through displacement of the newly synthesized strand from the donor template and its annealing with the 3Ј noninvading Received 10/16/02; accepted 4/8/03. single-stranded end to elicit GC only (Refs. 32–36; Fig. 1d). DSB The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with repair by GC occurs with fidelity and reflects the capacity of cells to 18 U.S.C. Section 1734 solely to indicate this fact. precisely restore the damaged DNA with no loss or gain of nucleotide 1 Supported by the National Cancer Institute of Canada for the Terry Fox Run, and in part by the Canadian Breast Cancer Research Initiative (to A. B.). A. B. is a scholar of the sequences (Fig. 1d). NHEJ differs from GC in that there is no Cancer Research Society/Medical Research Council of Canada, and of the Fonds de la requirement for extensive homology between DSB ends, and is po- recherche en sante´du Que´bec (FRSQ). J-F. V. and C. A. were the recipients of Canderel tentially error-prone, as nucleotide insertions and deletions are toler- fellowships. 2 To whom requests for reprints should be addressed, at CHUM-Hoˆpital Notre-Dame, ated at the rejoining sites. GC and NHEJ compete with one another Institut du Cancer de Montre´al, 1560, rue Sherbrooke est, Pavillon J.A. de Se`ve, Y-5634, (37), but very little is known about how eukaryotic cells elect one or Montre´al, Que´bec, Canada H2L 4M1. Phone: (514) 890-8000, extension 28946; Fax: the other (38). Under certain circumstances, eukaryotic cells can (514) 412-7591; E-mail: [email protected]. 3 The abbreviations used are: MMR, mismatch repair; HNPCC, hereditary nonpolypo- couple GC and NHEJ to seal DSBs and ensure their genome stability sis colorectal cancer; DSB, double-strand break; IDL, insertion/deletion loop; CMV, (Fig. 1e; Refs. 30, 32–34, 39–45). In these cases, GC intermediates cytomegalovirus; GC, gene conversion; PGC, precise gene conversion; NHEJ, nonho- mologous end joining; L1, long interspersed nuclear element; L1Md, long interspersed are interrupted at various stages and shunted to NHEJ, being charac- nuclear element of the mouse Musculus domesticus. terized by deletions, insertions, or duplications of DNA sequences at 3334 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2003 American Association for Cancer Research. ABERRANT DSB REPAIR ASSOCIATED WITH MSH2 DEFICIENCY single integration events were determined by Southern blot hybridization, using the Hyg gene as a probe. More than 80% of G418R colonies analyzed contained single pST100 recombinant molecules. For analysis of the recom- binants (Fig. 2), the following primer pairs were used in PCR: (a) Neo952 (5Ј-ccacgacgggcgttccttgcgcag-3Ј) and neo1300 (5Ј-gtcacgacgagatcctcgccgtc- 3Ј) amplify the 1.4 kb fragment, or 1.4 and 2.4 kb fragments, between the realigned neo cassettes; (b) Neo800 (5Ј-gaatagcctctccacccaag-3Ј) and hyg4419 (5Ј-gctgtgtagaagtactcgccg-3Ј) amplify the 2.1 kb fragment between the trans- ferred neo5Ј cassette and the Hyg gene; (c) Neo1300 and hyg4419 amplify the 3.6 kb fragment between the neo3Ј cassette and the Hyg gene; (d) Neo800 and pUC469 (5Ј-tgaccatgattacgccaagct-3Ј) amplify the 1.7 kb fragment between the neo5Ј cassette and pUC sequences; and (e) Neo1300 and pUC469 amplify the 3.2 kb fragment between the inverted neo3Ј cassette and pUC sequences Fig. 1. The one-sided invasion model proposed for DSB repair (32, 33). In this model, only 1 3Ј single-strand at a DSB (a) invades an intact homologous duplex (b) and primes new DNA synthesis to stabilize the intermediate (c). Resolution of such an intermediate can occur by unwinding of the newly synthesized strand, which can engage with the noninvading strand in annealing, leading to a PGC product (d), or in NHEJ, resulting in a rearranged product (e). When the newly synthesized strand is longer than the nonin- vading single-strand, NHEJ would lead to insertion/duplication at the joining site, whereas strand annealing would generate an intermediate with a protruding 3Ј tail (f). Such an intermediate can be resolved to a PGC product (d) or to a rearranged GC product (e)in the absence of stabilizing factors (see text). NHEJ junctions (30, 32–34, 39–46). Such rearranged GC products can be as frequent as PGC products (32–34, 39–41).
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