Copyright  1998 by the Society of America

Efficient Repair of All Types of Single-Base Mismatches in Recombination Intermediates in Chinese Hamster Ovary Cells: Competition Between Long-Patch and G-T -Mediated Repair of G-T Mismatches

Colin A. Bill,*,† Walter A. Duran,† Nathan R. Miselis* and Jac A. Nickoloff*,† *Department of Cancer Biology, Harvard University School of Public Health, Boston, Massachusetts 02115 and †Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 Manuscript received February 18, 1998 Accepted for publication April 27, 1998

ABSTRACT Repair of all 12 single-base mismatches in recombination intermediates was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks in regions of shared homology. Recombination was predicted to occur via single-strand annealing, yielding heteroduplex DNA (hDNA) with a single mismatch. Nicks were expected on opposite strands flanking hDNA, equidistant from the mismatch. Unlike studies of covalently closed artificial hDNA substrates, all mismatches were efficiently repaired, consistent with a nick-driven repair process. The average repair efficiency for all mispairs was 92%, with no significant differences among mispairs. There was significant strand-independent repair of G-T G-C, with a slightly greater bias in a CpG context. Repair of C-A was also biased (toward → C-G), but no A-C G-C bias was found, a possible sequence context effect. No other mismatches showed → evidence of biased repair, but among hetero-mismatches, the trend was toward retention of C or G vs. A or T. Repair of both T-T and G-T mismatches was much less efficient in mismatch repair-deficient cells -and the residual G-T repair was completely biased toward G-C. Our data indicate that single ,(%25ف) base mismatches in recombination intermediates are substrates for at least two competing repair systems.

ASE pair mismatches arise constantly in genomic nisms operate in both prokaryotes and (re- B DNA as a consequence of metabolic processes. Mis- viewed by Modrich 1991). There are three distinct mis- matches can result from incorrect base insertion, slip- match repair mechanisms in . One is long- page of DNA polymerase during replication, or strand patch repair mediated by mutHLS, which can recognize exchange during . The most single or multiple mismatches and excise a stretch, often frequent mismatch arises from spontaneous or geno- Ͼ1 kb, of newly synthesized DNA (reviewed by Rasmus- toxin-induced of either 5-methylcytosine, sen et al. 1998). In addition, there are two short-patch which accounts for 2–8% of all cytosine moieties repair pathways, involving vsr and mutY, that mediate (Doefler 1983; Riggs and Jones 1983), or cytosine to G-T G-C and G-A G-C repair, respectively (Lieb → → form G-T and G-U mismatches, respectively. The fre- 1983; Nghiem et al. 1988; Sohail et al. 1990; Tsai-Wu quency of these deamination processes is far greater et al. 1991). Although less well-characterized than in than the observed transversion expected for , a long-patch repair system has been suggested random repair of G-T or G-U mismatches (Shen et al. for Saccharomyces cerevisiae and mammalian cells because 1994) consistent with mechanisms that restore these to these have homologues of mutS and mutL (MSH and Brown Jiricny Schmutte G-C ( and 1987; et al. 1995). MLH)(Kramer et al. 1989b; Reenan and Kolodner Mismatch repair is necessary to reduce the accumula- 1992). There is also evidence for short-tract mismatch tion of mutations and to maintain genome integrity. In repair systems in eukaryotes. A thymine DNA glycosylase bacteria and yeast, mismatch repair-deficient cells show specific for G-T mismatches was identified in HeLa cell Levinson genome instability and a mutator phenotype ( extracts. This removes T in G-T mispairs, cor- Gutman Strand and 1987; et al. 1993), and in humans, recting to G-C (Wiebauer and Jiricny 1989, 1990). A mutations in mismatch repair are linked to the mammalian homologue of mutY (MYH) also has been genesis of certain cancers, including hereditary non- characterized (McGoldrick et al. 1995). polyposis colon cancer (Bronner et al. 1994; Boyer et Prolla In vertebrate cells the repair of all possible mispair- al. 1995; et al. 1998). In recent years, it has ed bases has been studied using either cell extracts become apparent that similar mismatch repair mecha- (Brooks et al. 1989; Holmes et al. 1990; Varlet et al. 1990; Thomas et al. 1991) or in vivo by transfection of DNA into cells containing mismatches created in vitro Corresponding author: Jac A. Nickoloff, Department of Molecular Folger Brown Jiricny Hey- Genetics and Microbiology, School of Medicine, University of New ( et al. 1985; and 1988; Mexico, Albuquerque, NM 87131. E-mail: [email protected] wood and Burke 1990). Although these studies indi-

Genetics 149: 1935–1943 (August 1998) 1936 C. A. Bill et al. cated that specific mismatch repair can occur, there was and increased mismatch segregation rates (Taghian a large variability in the overall efficiency and direction and Nickoloff 1998). Clone B cell extracts fail to com- of repair among different mismatch types. Several fac- plement extracts of LoVo cells, which are defective in tors may influence the direction and efficiency of mis- both alleles of hMSH2, and it was suggested that clone match repair. For example, an early study suggested B and LoVo share this defect (Aquilina et al. 1994). that status and DNA nicks were important Msh2-deficient mice also exhibit microsatellite instabil- determinants of mismatch repair strand discrimination ity, a mutator phenotype, and methylation tolerance in simian kidney cells (Hare and Taylor 1985). Many (de Wind et al. 1995). We show that repair of two mis- studies have since shown that DNA nicks increase the matches (T-T and G-T) is greatly reduced in clone B frequency of mismatch repair and bias repair toward cells, but residual repair of G-T still favored loss of T. the strand containing the nick (Holmes et al. 1990; These results indicate that single-base mismatches in Thomas et al. 1991; Umar et al. 1994; Miller et al. 1997; recombination intermediates are subject to repair by at Taghian and Nickoloff 1998). However, nick-directed least two distinct and competing mechanisms. repair may not be general; Heywood and Burke (1990) reported that nicks do not drive the direction of mis- MATERIALS AND METHODS match repair in simian COS-7 cells. Mismatch repair efficiency of particular mismatches is also influenced DNA constructions: were constructed by by the presence of other nearby mismatches, thought standard procedures (Sambrook et al. 1989). For transfection to reflect co-repair. Thus, a poorly repaired mismatch into CHO cells, highly supercoiled plasmid DNA was isolated by acidic-phenol extraction (Wang and Rossman 1994). can be repaired efficiently when located near a well- pneoAn was created by inserting the 2.1-kbp HindIII-BamHI repaired mismatch (Petes et al. 1991; Carraway and fragment of pSV2neo into the HindIII and BamHI sites of Marinus 1993; Weng and Nickoloff 1998). pUC19. Three single-base substitutions were created at posi- In a previous study we investigated mismatch repair tion 430 of the neo in pneoAn (Deng and Nickoloff Southern Berg in heteroduplex DNA (hDNA) formed by extrachromo- 1994) and pSV2neo ( and 1982) by unique- site elimination mutagenesis (Deng and Nickoloff 1994). somalrecombination between plasmid substrates in Chi- A frameshift was introduced into the resulting nese hamster ovary (CHO) cells (Deng and Nickoloff pSV2neo derivatives by insertion of a 10-bp XhoI linker (5Ј-CCC 1994). hDNA was predicted to be flanked by nicks on TCGAGGG-3Ј) into the BssHII site of neo to create four opposite strands. We found that two single-base mis- pSV2neoX(B) derivatives. Eight plasmids were used as recom- matches, located in close proximity, were repaired with bination substrates, with four plasmids each derived from pSV2neoX(B) and pneoAn containing each of the four possi- strong bias toward loss of the mismatched bases on the ble bases at position 430 in neo. An ApaI site was created when strand nearest to one of the flanking nicks. In two subse- the coding strand carried a C at position 430. quent investigations with substrates that produced three Cell culture and recombination assays: Wild-type (K1c) and to nine mismatches, all mismatch types tested were effi- mismatch-repair mutant (clone B) CHO cells were cultured Miller Taghian ciently repaired in CHO cells, and product spectra were as described previously ( et al. 1997; and Nickoloff 1998). For transfection experiments, derivatives consistent with most repair being directed from nicks of pSV2neoX(B) and pneoAn were linearized with MscI and (Miller et al. 1997; Taghian and Nickoloff 1998). BglII, respectively, and purified through Sepharose CL-6B Because these substrates produced hDNA with multiple (Nickoloff 1994). Electrotransformations of CHO cells were mismatches, the repair of each single-base mismatch was performed essentially as described by Taghian and Nicko- loff potentially influenced by the asymmetry of mismatch (1995), using the following conditions: 200 ng of each linearized plasmid was electroporated into a suspension of locations relative to flanking nicks and/or by co-repair 6 ϫ 106 cells in 0.75 ml of PBS, using a pulse of 300 V, of neighboring mismatches. The present study with indi- 960 ␮F and a 0.4-cm electrode gap. Plating efficiency for vidual mismatches located equidistant from flanking electroporated cells averaged 65% that of untreated controls. nicks was designed to investigate the repair of all types G418r recombinants were selected, and genomic DNA was of single-base mismatches in the absence of these con- prepared and used for plasmid rescue or PCR amplification as described (Taghian and Nickoloff 1998). Restriction founding factors. We report that all single-base mis- mapping of rescued plasmids and PCR products readily distin- matches are repaired with high efficiency. The majority guished recombinants from nonrecombinants (Taghian and of mismatches showed no repair bias, consistent with a Nickoloff 1998). nick-driven repair process. However, the repair of G-T The direction of repair of single-base mismatches was deter- mismatches was biased toward loss of T, and this bias mined either by digestion with ApaI or by a modification of an oligonucleotide hybridization procedure (Nickoloff and was strand-independent. Crosses were also performed Hallick 1982). Briefly, rescued plasmids or PCR products in mutant cells deficient in mismatch repair (“clone B”; were electrophoresed through duplicate 0.8% agarose gels Branch et al. 1993; Taghian and Nickoloff 1998). and transferred to Nytran membranes (Schleicher and Clone B cells share several characteristics with mutants Schuell, Keene, NH). Membranes were prehybridized for 1 hr m m defective in mismatch repair, such as tolerance to alkyl- in Church’s buffer (0.5 NaPO4, pH 7.2, 1 m EDTA, pH 8.0, 7% SDS; Church and Gilbert 1984) containing 100 ␮g/ ating agents, increased spontaneous mutagenesis and ml denatured salmon sperm DNA, and hybridized for 2 hr in microsatellite instability, defects in mismatch binding the same solution containing 32P-end-labeled DNA probe at a Kat Aquilina Hess ( et al. 1993; et al. 1994; et al. 1994), temperature 5Њ below the Tm of the probe. For each sample, Single-Base Mismatch Repair 1937 two hybridizations were performed with 15-base oligonucleo- tide probes (5Ј-TCTGTTGNGCCCAGT-3Ј, where N ϭ A, C, G, or T) complementary to each of the two possible repair outcomes of particular single-base mismatches. Membranes were washed in 2ϫ SSC (0.15 m NaCl, 0.015 m sodium citrate), 0.1% SDS, for 20 min each at 37, 42, and 49Њ and exposed to Kodak X-Omat AR film. In each case, one probe was com- pletely complementary to the target and the other had a mis- match at the central base; this mismatch destabilizes the hybrid and causes the probe to be washed off at lower stringency. By performing parallel hybridizations, we obtained positive and negative signals for each sample tested. Mismatch-repair effi- ciencies were measured using PCR-amplified DNA from prod- ucts with single, integrated recombinant molecules. Repaired recombinants hybridized to only one probe. Products that escaped repair segregated at the first round of replication after integration (producing a sectored colony) and hybridized to both probes. Repair data were analyzed by the StatXact-3 pro- gram (Statistical Solutions Limited) using a two-sided bino- Figure 1.—Structures of recombination substrates and mial distribution at the 95% confidence level. hDNA intermediates. pSV2neo and pneoAn derivatives were cleaved with MscI and BglII, respectively (top). Following trans- fection, these ends are processed to single-stranded regions, RESULTS exposing complementary strands (middle, left) that anneal to produce an hDNA intermediate with single-strand breaks Experimental design: The repair of single-base mis- in opposite strands (triangles) corresponding to DSB sites matches formed in vivo in hDNA intermediates by extra- (middle, right). The diagram shows the formation of the chromosomal recombination was studied with two types G-T mismatch. Following integration, recombinant molecules of plasmid recombination substrates as described pre- are rescued from genomic DNA by digesting with EcoRI, recir- Deng Nickoloff Miller cularizing with T4 DNA ligase, and transforming E. coli. Below viously ( and 1994; et al. 1997; are shown sequence contexts around position 430 in neo. In Taghian and Nickoloff 1998). pSV2neo derivatives the examples shown, the G-T mismatch mimics deamination contain a neo gene regulated by an SV40 promoter and of CpG to TpG (boxed), whereas the T-G mismatch mimics inactivated by a linker frameshift mutation. pneoAn de- deamination of CpC to TpC (boxed). rivatives contain an inactive neo gene because they lack a promoter (Figure 1). An SV40-driven neoϩ gene formed by recombination can integrate and produce G418r annealed plasmids would contain flaps with 3Ј ends; transfectants. Although extrachromosomal recombina- such flaps could be removed by homologs of yeast tion can occur by more than one mechanism; (De- Rad1p/10p and human ERCC1/4 (Ivanov and Haber sautels et al. 1990; Yang and Waldman 1992; see dis- 1995; Brookman et al. 1996). Greater digestion would cussion), single-strand annealing (SSA) (Lin et al. 1987, produce maximum hDNA flanked by single-stranded 1990; Carroll et al. 1994) is considered to be the pri- gaps that could be filled by extension of 3Ј ends. By mary recombination pathway. SSA might involve anneal- using this model to predict structures of recombination ing between single strands exposed by either a 5Ј to 3Ј intermediates, we performed 12 crosses to produce all exonuclease or a 3Ј to 5Ј exonuclease. Although an early types of single-base mismatches at position 430 of the report suggested 3Ј to 5Ј exonuclease activity in mouse neo gene. Because position 430 is essentially equidistant L cells (Lin et al. 1987), recent data indicate that ends from the two initiating breaks, markers at this position are processed by a 5Ј to 3Ј exonuclease in both mouse ES are expected to occur in hDNA at maximum rates. Data cells and yeast (Sun et al. 1991; Huang and Symington presented below indicate that mismatches were pro- 1993; Henderson and Simons 1997). Below we describe duced at position 430 at least 75% of the time. Mismatch genetic evidence for 5Ј to 3Ј exonuclease activity mediat- repair may occur before or after integration of the extra- ing SSA in CHO cells. chromosomal DNA into the genome. If a mismatch is Key predictions of the SSA model are that hDNA in not repaired, the resultant sectored colony can be iden- recombination intermediates may be flanked by nicks, tified by segregation analysis. Repair was analyzed in located on opposite strands corresponding to the re- wild-type (K1c) and mismatch repair-deficient mutant striction cut sites (Figure 1), and that a nonconservative (clone B; Branch et al. 1993) CHO cells. crossover product results. Depending on the extent of Silence of markers and recombination frequencies: exonuclease digestion, SSA intermediates may include The point mutations introduced into position 430 of more or less hDNA in the region between the double- neo were at a codon third position and were expected strand breaks (DSBs), with maximum hDNA (242 bp) to maintain the neo sequence. The silence formed if exonuclease digestion extends from broken of these mutations was confirmed by electroporating ends to (or past) DSB positions in recombining part- pSV2neo derivatives into CHO cells and selecting for ners. Lesser digestion would produce less hDNA and G418-resistant colonies, and by transformation of E. coli 1938 C. A. Bill et al.

TABLE 1 Recombination frequencies in wild-type and mutant CHO cells

Mismatcha Strain Frequency (ϫ 106)b nc A-A K1c 4.5 Ϯ 1.8 3 C-C K1c 5.6 Ϯ 3.3 5 Figure G-G K1c 3.3 Ϯ 0.8 3 2.—Single-base marker detection by hybridizing T-T K1c 11.1 Ϯ 2.5 3 mismatched/matched oligonucleotide probes. Eleven res- A-C K1c 9.5 Ϯ 6.2 3 cued recombination products from a T-C mismatch cross were run in duplicate on 0.8% agarose gels, blotted and hybridized C-A K1c 4.7 Ϯ 0.8 3 with 15-base oligonucleotide probes that match either the A-G K1c 6.5 Ϯ 2.3 3 G-C form (G) or the T-A form (T). Autoradiograph shows G-A K1c 3.0 Ϯ 1.3 3 supercoiled and relaxed circular plasmids. See materials and Ϯ C-T K1c 8.5 1.3 3 methods for hybridization details. T-C K1c 5.5 Ϯ 1.3 3 G-T K1c 11.0 Ϯ 1.4 3 T-G K1c 3.8 Ϯ 0.9 4 G-T clone B 9.6 Ϯ 3.0 5 consistent with repair directed from the proximal nick T-T clone B 10.3 Ϯ 1.9 5 (Deng and Nickoloff 1994). Similarly, two studies with multiple mismatches suggested that repair direction for a First base listed is on the top strand, donated by pSV2neo derivative. various mismatches was generally proportional to their b Recombination frequencies were calculated as the ratio distance from DNA nicks (Miller et al. 1997; Taghian of G418r colonies per viable cell electroporated. Values are and Nickoloff 1998). In the present study the single- averages ϮSD. From 0.05 to 0.2 ␮g of each DNA was used base mismatches are predicted to be 130 bp and 112 bp in transfections; all frequencies are normalized to 0.1 ␮gof from nicks on the top and bottom strands, respectively. pSV2neo derivative. c Number of repetitions. Therefore, if all repair was nick-directed, a mismatched base on the top strand, located 54% of the distance from the nick on the top strand [130 bp ϭ 0.54 (130 with derivatives of pSV2neo or pneoAn and selecting bp ϩ 112 bp)], would on average be retained 54% of for kanr colonies. Each of the six mutant plasmids and the time (or lost 46% of the time). We plotted the data the two wild-type plasmids reproducibly yielded similar from Table 2 relative to the value expected if repair was numbers of G418r or kanr colonies (data not shown). solely determined by nicks (Figure 3). For three crosses Recombination frequencies in CHO K1c cells were there were significant departures from the value pre- 5–10 ϫ 10Ϫ6/0.1 ␮g of plasmid DNA for each of 12 dicted for nick-directed repair. One pair of crosses dis- crosses (Table 1). Recombination frequencies for G-T played strand-independent biased repair: those pre- and T-T crosses, measured in parallel in K1c and clone dicted to yield G-T and T-G mismatches (assuming SSA B cells to control for variation in the input plasmid mediated by a 5Ј to 3Ј exonuclease) were significantly , gave similar values (Table 1). These recombina- biased toward G-C (P ϭ 0.016) and C-G (P ϭ 0.035), tion frequencies are Ͼ100-fold above the background respectively. For combined G-T/T-G data, P ϭ 0.01, levels determined by electroporation of individual indicating a clear strand-independent bias toward loss pSV2neoX(B) or pneoAn plasmids (data not shown), of T. Different mismatches are predicted for different as seen previously (Taghian and Nickoloff 1998). exonuclease polarities, so the crosses predicted to yield Evidence for mismatch-specific and nick-directed re- A-C and C-A with a 5Ј to 3Ј exonuclease would yield pair: G418r colonies were isolated for each of 12 crosses, G-T and T-G, respectively, with a 3Ј to 5Ј exonuclease. and recombinant products were rescued. We found that However, of this pair, only C-A displayed a significant Ͼ70% of rescued plasmids had the structure expected repair bias (P ϭ 0.003); no bias was seen with A-C from a nonconservative exchange between pSV2 (P ϭ 0.597). The bias in C-A repair may reflect a se- neoX(B) and pneoAn. Mismatch-repair direction was quence context-dependent C-A binding activity (see dis- scored in rescued plasmids by ApaI digestion or by oligo- cussion). Thus, strand-independent repair bias was ob- hybridization, an example of which is shown served only with that pair of crosses predicted to yield in Figure 2. An average of 25 products with correct G-T mismatches with a 5Ј to 3Ј exonuclease. This bias structures was analyzed per cross to determine repair is consistent with the well-known G-T G-C repair → direction for each type of mismatch. Segregation analy- systems in bacteria (Sohail et al. 1990; Lieb 1991) and in sis (described below) also yielded information about mammalian cells (Brown and Jiricny 1988; Wiebauer repair direction. The combined data from these two and Jiricny 1989, 1990). These data provide genetic approaches are shown in Table 2. evidence for a 5Ј to 3Ј exonuclease and also suggest that In a previous study, mismatches located asymmetri- both mismatch-specific and nick-directed repair con- cally between predicted nicks were repaired with bias tribute to the observed product spectra. Single-Base Mismatch Repair 1939

TABLE 2 Direction and efficiency of single-base mismatch repair in CHO K1c cells

No. of products repaired to:b Segregation analysis Mismatcha A-T T-A C-G G-C No. tested % Repair A-A 23 20 — — 10 100 C-C — — 15 12 12 83 G-G — — 12 18 9 89 T-T 25 20 — — 11 91 A-C 21 — — 22 10 90 C-A — 6 26 — 10 100 A-G 17 — 24 — 21 95 G-A — 16 — 22 14 100 C-T 11 — 21 — 10 80 T-C — 23 — 21 11 91 G-T 9 — — 27 13 92 T-G — 14 25 — 13 85 a First base listed is on top strand, donated by pSV2neo derivative. b Cumulative data obtained from rescued products and from segregation analysis.

All single-base mismatches are repaired efficiently in for all mispairs was 8.3%, with no significant differences wild-type cells: A prior study indicated that markers among mispairs. These efficiencies are quite high com- located 349–523 bases from flanking nicks occur in pared to those seen with closed circular DNA (Brown hDNA Ն50% of the time (Taghian and Nickoloff and Jiricny 1988), as discussed below. 1998). We therefore expected that the position 430 Although efficient repair is one explanation for the markers, located only 130 bases or less from flanking low segregation rates seen in CHO K1c cells, some re- nicks, would occur in hDNA at even higher rates (see combinants may form without position 430 being in- below). Segregation analysis indicates that all mis- cluded in hDNA and would be incorrectly scored as matches are repaired at high efficiency in wild-type “repaired.” To investigate this possibility, we performed CHO K1c cells (Table 2). The mean rate of segregation two crosses in mismatch repair-deficient clone B cells predicted to produce T-T and G-T mismatches. In con- %75ف ,trast to the Ͻ10% segregation in CHO K1c cells segregation was observed in the mutant clone B cells (Figure 4). Interestingly, the nonsegregating T-T mis- match products were evenly distributed (three products corrected to A-T, two corrected to T-A), whereas seven of seven nonsegregating G-T mismatch products carried G-C pairs at position 430. The observation that G-T repair efficiency is reduced, and yet retains significant bias, provides insight into the competition between pro- teins involved in nick-directed and G-T-specific mis- match repair (see below).

DISCUSSION Most extrachromosomal recombination in mamma- lian cells is thought to proceed by a nonconservative Figure 3.—Percentage retention of the base on the top mechanism that yields apparent crossover products strand [listed first in each mismatch; donated by pSV2- Seidman Lin neoX(B)]. The horizontal line at 54% retention represents ( 1987; et al. 1990). Such products in theory the predicted value if all mismatch repair were nick-directed. could arise by exchange in which hDNA forms infre- Data are from Table 2. Values significantly different from the quently and/or is limited in extent (Desautels et al. nick-directed model (P Ͻ 0.05, binomial confidence limits) 1990), or via intermediates with extensive hDNA formed are marked with asterisks. Data for the eight hetero-mis- by SSA as seen in three prior studies (Deng and Nicko- matches are shown on the right, with solid circles showing loff Miller Taghian Nickoloff repair favoring either C or G and open circles showing repair 1994; et al. 1997; and favoring A or T. Homo-mismatches are indicated by shaded 1998). This study provides two lines of evidence for circles. frequent hDNA. First, three crosses (C-A, G-T, and T-G; 1940 C. A. Bill et al.

mismatched bases (Figure 3), as seen with circular sub- strates in monkey cells (Brown and Jiricny 1988). We observed differential repair biases of C-A and A-C mismatches. C-A was preferentially repaired to C-G (81%), and A-C G-C showed no bias (Figure 3). In our → system, changing the orientation of hetero-mismatches changes the sequence context (Figure 1). C-A-specific repair may involve a protein analogous to a human protein that displays context-sensitive binding to C-A mismatches. The C-A binding protein recognized C-A mismatches in an ApG context, but not an ApA context (italics denote mismatched base; O’Regan et al. 1996). Consistent with this, the C-A repair bias was seen in an ApG context, whereas no bias was seen for the A-C mismatch in an ApC context. Thus, the enzyme may only recognize C-A mismatches in the ApG context (ApT has not yet been tested). O’Regan et al. (1996) sug- Figure 4.—Percentage repair of G-T and T-T mismatches gested that sequence context-sensitive mismatch-spe- in wild-type K1c and mismatch repair-deficient clone B cells. cific repair systems form a complementary repair net- Data for K1c cells are from Table 2. G-T and T-T data for work. Sequence context effects may reflect different clone B cells are from 26 and 24 products, respectively. Seven of seven G-T mismatches repaired in clone B cells were cor- thermodynamic stabilities and helix melting enthalpies rected to G-C. of mismatched bases (Aboul-ela et al. 1985; Werntges et al. 1986). Differential repair may also result from transcriptional asymmetry. In bacteria, repair efficienc- Figure 3) exhibited marker retention/loss rates that ies of mutY-dependent repair of G-A and C-A mismatches deviated from values expected if hDNA did not form. to G-C and C-G, respectively, were influenced by both Second, segregation analysis in clone B cells indicated sequence context and transcription (Fox et al. 1994). hDNA forms at least 75% ofthe time (Figure 4). Further- The most prevalent mismatch is G-T, arising by deami- more, although 25% of the nonsegregating products nation of 5-methylcytosine. Several studies in eukaryotic might have arisen in the absence of hDNA, one would systems have demonstrated biased G-T G-C repair expect that in this case the centrally located marker (Brown and Jiricny 1988; Heywood and→Burke 1990; would be evenly distributed between the two parental Holmes et al. 1990) showing little strand bias (Brown configurations. Instead, seven of seven nonsegregating and Jiricny 1987; Holmes et al. 1990), although such G-T products in clone B cells had G-C base pairs. The a repair proclivity was not always seen (Varlet et al. probability of obtaining this result in the absence of 1990). G-T-specific thymine glycosylase is known to me- hDNA is very low (P ϭ 0.57 ϫ 2 ϭ 0.016), suggesting diate G-T G-C repair (Wiebauer and Jiricny 1990; → that most or all of these products arose by biased repair Neddermann and Jiricny 1993; Gallinari et al. 1998). of hDNA (likely mediated by G-T glycosylase; see below) In vertebrates, 5-methylcytosine typically occurs in CpG and that SSA produces hDNA at position 430 at rates dinucleotides. Human cell extracts and purified human approaching 100%. G-T glycosylase recognize G-T mismatches most effi- In E. coli, G-A mispairs are repaired to G-C by the ciently in a CpG context (Griffin and Karran 1993; MutY adenine-specific DNA glycosylase (Au et al. 1989). Sibghat-Ullah and Day 1995; Sibghat-Ullah et al. The identification of a human mutY homologue (MYH) 1996), consistent with a role for G-T glycosylase in coun- suggests that a similar mechanism may operate in mam- teracting G-T mismatches in this context. Similar con- malian cells (McGoldrick et al. 1995). Biased G-A to text effects may modulate G-T processing in recombina- G-C repair was observed in circular substrates (Brown tion intermediates in vivo. We found somewhat greater and Jiricny 1988) and recombination intermediates bias for G-T in a CpG context (75%) than for T-G in a (Miller et al. 1997), but no G-A or A-G repair biases CpC context (64%; Figure 1), although this difference were found in this study, possibly reflecting sequence was not statistically significant with these sample sizes. context effects (see below). The mammalian all-type The purified G-T glycosylase also recognizes and re- endonuclease can nick all single-base mismatches with moves thymine from other mismatches, including T-T, different efficiencies (Yeh et al. 1991), but its function but at a lower efficiency (Neddermann and Jiricny is unclear, and it may not be involved in mismatch cor- 1993, 1994). The residual repair of T-T in clone B cells rection (Gallinari et al. 1998). Although we found (Figure 4) may therefore be mediated by the G-T glyco- most single-base mismatches were repaired without sig- sylase as well. nificant bias, there was a general trend toward retention With at least two systems capable of acting on G-T of either C or G vs. A or T in the four pairs of hetero- mismatches, the question arises whether the systems are Single-Base Mismatch Repair 1941 in competition. Gallinari et al. (1998) argued that and nicked DNA, but our data are nevertheless consis- G-T mismatches arising by deamination are not pro- tent with the idea that nicks enhance repair because cessed by a nick-directed pathway (i.e., postreplication our 8% average segregation rate is fourfold lower than mismatch repair) because it is unlikely that nicks will the average for closed circular DNA (Brown and Jir- occur near sites of deamination. They further argued icny 1988). Thus, studies with closed circular DNA, or that G-T-specific repair does not interfere with nick- singly nicked substrates, demonstrate that repair effi- directed repair of G-T mismatches formed by replication ciency varies among mismatch types and that repair errors. In contrast, our data suggest that nick-directed direction is influenced by which strand contains a nick, and G-T-specific repair do compete during processing whereas our data indicate that flanking nicks in recom- of recombination intermediates. Our observed 64 and bination intermediates direct efficient repair of all sin- 75% biases toward G-C for the two G-T crosses are lower gle-base mismatches, including those found to be poorly than the 85 and 93% biases seen with circular DNA repaired in the absence of nicks, such as T-T and A-G. (Brown and Jiricny 1987), consistent with the idea Long-patch mismatch repair in bacteria involves (1) that nick-directed repair reduces bias imposed by G-T- mismatch binding by MutS; (2) signaling from the specific repair. The completely biased (albeit low-effi- bound mismatch to MutH (possibly involving MutL); ciency) repair of G-T G-C in clone B cells lends (3) nicking at a hemi-methylated GATC site by MutH; → further support to this idea. This low-efficiency repair and (4) excision repair directed from the nick. Al- would appear to be in conflict with the observation that though certain mismatches frequently escape repair in these cells lack detectable G-T binding activity (Branch E. coli and yeast, such as C-C (Bishop et al. 1989; Kramer et al. 1993). However, clone B cells presumably lack et al. 1989a; Lichten et al. 1990; Detloff et al. 1991) Msh2 (Aquilina et al. 1994), and most G-T binding may and palindromic loop mismatches (Nag et al. 1989; Nag be Msh-dependent as these proteins were reported to and Petes 1991; Weng and Nickoloff 1998), recent be more abundant and bind more efficiently to G-T studies in yeast indicate that, at least for palindromic mismatches than G-T glycosylase (Sibghat-Ullah et al. loop mismatches, poor repair is not due to inefficient 1996). Previously, we concluded that biased repair of mismatch recognition (Moore et al. 1988; Nag et al. palindromic loop mismatches is also reduced by nick- 1989; Alani 1996; Manivasakam et al. 1996). For exam- directed repair. Interestingly, palindromic loop repair ple, although single-base and palindromic loop mis- is highly efficient in both wild-type and clone B cells matches are both bound by yeast Msh2p-Msh6p (Alani (Taghian and Nickoloff 1998), whereas G-T repair 1996), binding is modulated differently by adenosine in clone B cells is reduced to 27% of wild-type levels triphosphate, providing a potential clue as to why these (Figure 4). Thus, G-T and loop mismatches are repaired are differentially repaired. If mismatch repair in mam- by distinct mismatch-specific systems, both of which malian cells parallels that in yeast and E. coli, then the compete with nick-directed repair in recombination in- different repair efficiencies seen in nicked and non- termediates. nicked substrates in mammalian cells may not reflect A key prediction of the SSA model is the presence differences in mismatch binding. Instead, the rate-lim- of nicks (or single-stranded gaps) on opposite strands iting step may involve signaling to a nicking enzyme. flanking hDNA in recombination intermediates. Previ- Our data suggest that when nicks are provided, all mis- ous studies of such intermediates with multiple mis- matches are repaired efficiently. matches indicated that repair was highly efficient We thank E. Miller, D. Taghian, and E. Alani for helpful com- (Miller et al. 1997; Taghian and Nickoloff 1998). ments. This research was supported by grant CA-54079 to J. Nickoloff However, these studies could not distinguish whether from the National Cancer Institute, National Institutes of Health. repair efficiencies of particular mismatches were influ- enced by co-repair at adjacent mismatches. We now report an overall repair efficiency of 92% for all types LITERATURE CITED of individual single-base mismatches in recombination Aboul-ela, F., D. Koh I. 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