Copyright © 1998 by the Genetics Society of America
Genetics 148: 59–70 (January, 1998)
Evidence for Independent Mismatch Repair Processing on Opposite
Sides of a Double-Strand Break in
Saccharomyces cerevisiae
Yi-shin Weng and Jac A. Nickoloff
Department of Cancer Biology, Harvard University School of Public Health, Boston, Massachusetts 02115 Manuscript received August 8, 1997
Accepted for publication September 26, 1997
A B S T R A C T
Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT lo-cus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape re-pair, and segregate to produce a sectored (Ura1/2) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palin-drome, and a 37-bp near-palinpalin-drome, z75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these tional markers increased loop repair (likely reflecting corepair). Among sectored products, few had addi-tional segregating markers, indicating that the lack of repair at one marker is not associated with ineffi-cient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than non-sectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.
elson and Radding 1975). In this view, the
predomi-nance of gene conversion over PMS reflects efficient mismatch repair. The gap repair model was proposed to explain the results of transformation experiments in which an allele containing a double-strand gap (pro-duced in vitro) was repaired from endogenous sequences in the host genome (Orr-Weaveret al. 1981; Szostak
et al. 1983). In this model, most conversion occurs in a double-strand gap, which would preclude PMS, but rare PMS events were thought to reflect the inclusion of markers in heteroduplex DNA (hDNA) adjacent to a gap during strand invasion or as a result of Holliday junction (HJ) branch migration.
In both mitotic and meiotic cells, nearly all conver-sion tracts are continuous (Aguilera and Klein 1989; Ahn and Livingston 1986; Borts and Haber 1987;
Borts and Haber 1989; Judd and Petes 1988;
Sweet-seret al. 1994; Symington and Petes 1988; Willis and Klein 1987), and alleles suffering a DSB are
preferen-tially converted (reviewed in Nickoloff and Hoek-stra 1997). These observations, plus the fact that
gapped substrates are repaired in vivo with information donated by a homologous duplex (Orr-Weaver et al. 1981), provided strong support for the gap repair model. However, in vivo studies of MAT conversion failed to Corresponding author: Jac A. Nickoloff, Department of Molecular
Genetics and Microbiology, School of Medicine, University of New Mexico, Albuquerque, NM 87131. E-mail: [email protected]
G
ENE conversion is the nonreciprocal transfer of information from a DNA duplex to a homolo-gous duplex, a process that has been widely studied in yeast (reviewed in Peteset al. 1991). For heterozygous loci, gene conversion results in a 3:1 (or 6:2) aberrant meiotic segregation pattern, contrasting with normal 2:2 segregation. In the yeast Saccharomyces cerevisiae, sev-eral other types of aberrant segregation patterns are observed at lower frequencies, notably 5:3 patterns, in which one of the four spores yields a sectored colony. Sectoring reflects segregation of mismatches, which are formed during strand exchange, in the mitotic division immediately following meiosis. These 5:3 patterns are therefore termed postmeiotic segregation (PMS).Mes-60 Y.-s. Weng and J. A. Nickoloff
show double-strand gap formation at double-strand breaks (DSBs) introduced into MAT by HO nuclease (White and Haber 1990), and genetic evidence sug-gests that markers quite close to the MAT DSB are of-ten in hDNA (Haber et al. 1993; McGillet al. 1989; Ray et al. 1991). Furthermore, although meiotic con-version is initiated by DSBs (Caoet al. 1990; de Massy
et al. 1995; Nag and Petes 1993; Sunet al. 1989; Wu and Lichten 1994), PMS increases in mismatch repair-defective strains to the same degree that gene conversion decreases, suggesting that most or all meiotic conver-sion results from mismatch repair of hDNA (reviewed in Peteset al. 1991). Direct evidence for hDNA mediat-ing meiotic gene conversion was obtained in two stud-ies (Lichtenet al. 1990; Nag and Petes 1990; Nag and Petes 1993). To account for tract continuity and
pref-erential conversion of broken alleles in an hDNA re-pair model, mismatch rere-pair must involve long rere-pair tracts (or less likely, concerted short repair tracts), and repair must be biased against mismatched bases in re-cipient alleles (i.e., those suffering a DSB). E. coli has systems for both long and short tract mismatch repair (Modrich 1991). The long tract repair system involves MutHLS, and yeast has MutL and MutS homologs (re-viewed in Crouse 1997). Specialized short tract repair systems are unknown in S. cerevisiae, but Schizosaccharo-myces pombe apparently has a system that repairs C–C mismatches (Schar and Kohli 1993). Previous studies showed that for spontaneous mitotic conversion, sec-tored colonies and discontinuous conversion tracts arise at frequencies of z15% and z3%, respectively (Ronne and Rothstein 1988; Sweetseret al. 1994). Although these results indicate that at least some mitotic conver-sions involve hDNA intermediates, they do not differ-entiate between hDNA and gap repair models for the majority of mitotic events. Indirect evidence against gap repair for general mitotic DSB-induced conversion (vs. site-specific conversion at MAT) comes from two studies involving conversion of plasmid-borne alleles interacting with chromosomal loci. In these studies, conversion tracts often extended in only one direction from a DSB (or gap) (Nelsonet al. 1996; Priebe et al. 1994). This result is inconsistent with gap repair since gaps are unlikely to be formed on only one side of a DSB. In the present study we addressed this question directly by using palindromic frameshift insertion mu-tations adjacent to DSB sites that would produce poorly repaired loop mismatches if present in hDNA, an ex-periment suggested by Esposito et al. (1994). Segre-gation of loop mismatches produces sectored (1/2) colonies, which were seen at a frequency of z75% among recombinant products, indicating that most DSB-induced mitotic conversion is mediated by hDNA repair. The system was then exploited to examine the fate of additional mismatches near an unrepaired loop mismatch. These experiments indicated that the addi-tional mismatches are efficiently repaired despite their
proximity to the unrepaired loop mismatch and that conversion on opposite sides of a DSB can be mediated by two independent mismatch repair tracts.
M AT E R I A L S A N D M E T H O D S
Plasmids: Standard techniques were used to construct plas-mids (Sambrooket al. 1989). Plasmid pUCUraR-HO432D59Hleu is a pUC19 derivative with ura3 and LEU2 (J. W. Cho and J. A. Nickoloff, unpublished results). This plasmid contains ura3 with nine phenotypically silent RFLP markers and a 24-bp HO site at a natural NcoI site at position 432 (HO432) (Sweetser et al. 1994). This plasmid lacks the polylinker XbaI site, and the promoter proximal (59) HindIII site of the ura3 fragment was deleted to facilitate allele rescue and simplify marker analysis. Derivatives of this plasmid were created as follows. A 14-bp palindromic frameshift mutation was created by insert-ing a 10-bp MluI linker (59-CGACGCGTCG-39) into the silent PmlI RFLP at position 409 (Pml409). Subsequent MluI diges-tion, fill-in, and ligation reactions converted the MluI site to a BssHII site, and created a palindromic 14-bp insertion (Bss14-409). An 8-bp NruI linker (59-GTCGCGAC-39) was inserted into a digested and filled-in Bss14-409 site, and a 12-bp MluI linker (59-GCGACGCGTCGC-39) was then inserted into the NruI site. Although the resulting insertion carried the desired MluI site, sequence analysis showed that it did not have the predicted 38-bp sequence; one bp was absent, an apparent cloning artifact. This 37-bp near-palindromic insertion is called Mlu37-409. The HO site and either Bss14-409 or Mlu37-409 were transferred to a wild-type copy of URA3 in a pUC19 de-rivative lacking the polylinker XbaI site by using a domain re-placement procedure (Ray et al. 1994).
Yeast strains: Cells were cultured as described previously (Nickoloff et al. 1990). Strain DY3421 (J. W. Cho and J. A. Nickoloff, unpublished results) contains ura3 with a 11 frameshift mutation at position 764 (X764) (Sweetser et al. 1994) and a galactose-inducible source of HO (GalHO) inte-grated at lys2 using pHSS19, a kanamycin-resistant vector (Nickoloff and Reynolds 1991); this vector does not inter-fere with rescue of either ura3 allele, which are linked to the ampicillin-resistant pUC19 vector. DY3421 also carries a MATa-inc mutation to prevent mating-type switching when HO is in-duced, ade2-101, his3-200, leu2-D1, and trp1-D1 (Sweetser et al. 1994). Plasmids with ura3 containing 14- or 37-bp palin-dromes and LEU2 were linearized at the SmaI site in ura3 and integrated into ura3-X764 of DY3421, creating strains YW14-409 and YW37-YW14-409. Integration of a related plasmid contain-ing a 14-bp palindrome and 8 silent RFLP markers created strain YW14-409R. Structures of the resulting duplications (Figure 1, A and B) were confirmed by Southern hybridiza-tion analysis of genomic DNA. Further characterizahybridiza-tion of res-cued ura3 alleles (see below) included restriction mapping of all markers, and sequence analysis of the region containing palindromes, including the entire region amplified by PCR during domain replacement.
identi-fied by replica-plating to uracil omission medium. LB-Ura me-dium is identical to standard uracil omission meme-dium except that it contains 3.4 g/liter yeast extract, 6.8 g/liter bacto-tryp-tone, and 3.4 g/liter NaCl. LB-Ura contains enough uracil to support growth of Ura2 yeast, but allows the Ura phenotype to be determined visually in ade2 (red) mutants (Weng and Nickoloff 1997). On LB-Ura plates, three types of colonies were identified: white (Ura2 parental), red (Ura1 nonsec-tored recombinants), and half-red/half-white (Ura1/2 sec-tored recombinants). Leu phenotypes were subsequently de-termined for all recombinants, including both halves of sectored colonies. Strain YW14-409R was treated as above except that HO was induced for 6 hr. Although increasing the length of induction would tend to reduce the ratio of sectored to non-sectored products due to preplating segregation, preliminary tests indicated that the longer induction increased the total number of both sectored and nonsectored recombinants. This simplified the recovery of sectored colonies, which arose at very low frequencies in YW14-409R (see results), and did not compromise our physical analysis of sectored products.
Sectored product analysis: Conversion tracts, and segre-gated hybrid DNA regions in sectored products of strain YW14-409R were characterized by mapping both alleles from Ura1 and Ura2 sectors. This physical analysis was performed only on sectored colonies in which both sectors were Leu1, as these were constrained to arise by gene conversion (Ura1/2 sectors can also result from unequal sister chromatid
pillae; strains lacking HO sites (i.e., those in which the HO site had converted) recombine infrequently and few or no pa-pillae arise. Statistics were performed using the Fisher exact test.
R E S U LT S
Experimental strategy: To determine whether
gen-eral, DSB-induced gene conversion is mediated by gap or hDNA repair, we constructed strains with nontan-dem ura3 duplications having perfect or near perfect palindromes 23 bp from an HO site. DSBs can be deliv-ered to HO sites in vivo, thus providing a defined re-combination initiation site (Figure 1). We reasoned that if DSB-induced gene conversion involves even short double-stranded gaps, the palindrome would fre-quently be included in gaps extending from the nearby HO site, and Ura1 recombinants would usually be non-sectored. However, if conversion proceeds by hDNA re-pair, as seen in meiosis and during MAT switching (Nag and Petes 1990; Nag and Petes 1993; Petes et al. 1991; Ray et al. 1991), the palindrome should produce a stem-loop mismatch (Figure 1, C and D) that is ex-pected to frequently escape repair (Nag and Petes 1991; Nag et al. 1989) and segregate during the next di-vision, producing sectored Ura1/2 recombinants. Since sectored colonies will only be detected if recombinants do not divide before plating, recombination was in-duced only briefly (3 hr in galactose medium). Cells grown in galactose divide about once per 2–3 hr (data not shown), so these conditions allow no more than one cell division during the induction period. Unin-duced recombination frequencies were less than 1024 (data not shown) while induced frequencies were z2 3 1021 (Table 1), confirming that essentially all recombi-nants from galactose cultures were induced by DSBs.
DSB-induced recombination between these ura3 di-rect repeats can produce a variety of products includ-ing full or half gene conversions (Ura1 or Ura1/2) in
which both ura3 repeats and LEU2 are retained (Leu1 or Leu1/1) or that are associated with a deletion of one ura3 repeat and LEU2 (Leu2 or Leu2/2). Deletions may reflect either crossover or single-strand annealing (SSA) events. Also expected were unequal sister chro-matid exchange events (which produce a colony with a Leu1 triplication sector and a Leu2 deletion sector). If recombinants are identified using selective medium, gene conversions and triplications yield identical Ura1 Leu1 phenotypes, but can be distinguished if genomic DNA is analyzed. Deletions arising by crossing-over, SSA, or unequal exchange are indistinguishable pheno-typically (all are Leu2) and genotypically. The nonse-lective conditions used in this study allow sectored gene conversion products (Ura1/2 Leu1/1) to be easily dis-tinguished from other types of Ura1/2 sectored products, such as triplication or deletion events resulting from unequal sister chromatid exchange (Ura1/2 Leu2/1 and Ura1/2 Leu1/2) (Figure 2).
Distribution of recombinant products: The phenotype distribution for DSB-induced recombinants of strain YW14-409 is shown in Table 1. Ura1/2 Leu2/2 deletion products appeared most frequently; these may arise by loop segregation during conversion events associated with a crossover or an SSA event, or from conversion of only one sister chromatid in G2 cells, although evi-dence presented below argues against the latter mecha-nism in the majority of cases. Leu1/1 gene conversion (non-deletion) products were less frequent than dele-tions (Leu2/2), consistent with studies using related strains (Nickoloff et al. 1989; J. W. Cho and J. A. Nickoloff, unpublished results). Leu1/2 products ac-counted for z40% of the total recombinants. Southern analysis of three Leu1/2 products each of classes 5 and 6 (Figure 2) was consistent with these resulting from unequal sister chromatid exchange or by the mecha-nism proposed by Lovett et al. (1993) involving sister strand exchange of nascent strands during DNA
repli-TABLE 1
DSB-induced recombination frequencies
Strain na
1043 Frequencyb
Full GCc Half GC
Percent Sectored
Deletion 6 GC Deletion 1 Half GC
Deletion/Triplicationd
Ura1/2 Leu2/1
Ura1/2 Leu1/2
Ura1/1 Leu1/2 Ura1 Leu1 Ura1/2 Leu1/1 Ura1 Leu2 Ura1/2 Leu2/2
YW14-409 9,205 38 265 87 170 798 425 425 44
YW37-409 9,789 90 427 83 150 682 256 341 19
YW14-409R 17,167 253 12 5 ND 67 17 16 ND
ND 5 not determined. a Number of colonies analyzed.
b Frequencies of DSB-induced products with indicated phenotypes. See Figure 2 for chromosomal corresponding to each col-ony phenotype.
c GC 5 gene conversion.
cation (G2 events), as Leu1 and Leu2 halves had ura3 triplications and single copies of ura3, respectively (data not shown). Thus, most Leu1/2 products proba-bly reflect sister chromatid interactions, but this limited analysis may have missed products arising via deletion in only one sister chromatid in G2. This triplication fre-quency is considerably higher than seen in a study of a limited number of products with a related cross ( Nick-oloff et al. 1989). However in that study, recombinants
were identified following selection. Of the three tripli-cation classes shown in Figure 2, only classes 6 and 7 would be scored as unequal exchange if only Ura1 products were recovered due to selection, reducing this fraction to z20% of the total. The Ura2/2 Leu2/2 dele-tion class (expected to be common, see ref. Nickoloff
et al. 1989) and the fourth unequal exchange class
(Ura2/2 Leu1/2) were not scored. In the present study deletions and unequal exchange products were not
characterized extensively because of ambiguities associ-ated with their formation (e.g., deletions may result from reciprocal exchange or SSA). Instead, we focused on the more mechanistically constrained conversions unassociated with deletions (Leu1/1; Figure 2, classes 1 and 2) because our primary goal was to investigate the mechanism(s) of DSB-induced gene conversion. Simi-lar product distributions were found for strains YW14-409 and YW37-YW14-409 (Table 1), indicating that the 37-base loop is also poorly recognized by the mismatch repair machinery and that loop length does not affect recom-bination mechanism(s).
Most DSB-induced mitotic gene conversion is
mediat-ed by hDNA repair: Among YW14-409 and YW37-409
products that remained Leu1, z87% were sectored Ura1/2. To confirm that these arose via segregation of a palindromic mismatch, and not from conversion of only one sister chromatid in G2, we analyzed Ura2 sec-tors from strain YW14-409. If such secsec-tors arose from conversion of only one sister chromatid in G2 cells, they would retain the HO site and would recombine at high frequency upon growth in medium with galactose. Patch recombination assays (see materials and meth-ods) on 17 Ura2 sectors from Ura1/2 Leu1/1 products showed that 4 retained an HO site. Therefore, the ma-jority of the Ura1/2 Leu1/1 sectors reflect the segrega-tion of the palindromic frameshift mutasegrega-tion in hDNA. Because a significant fraction of events occur in G2 (discussed above), these data suggest that the majority G2 events involve cleavage of HO sites in both sister chromatids; products with quarter-sectors were appar-ent on LB-ura medium, but these were z10-fold less frequent than half-sectors, further supporting this idea. Physical analysis of 22 Ura1/2 Leu1/1 sectored prod-ucts from the related strain YW14-409R (see below) in-dicated that none resulted from conversion of only one sister chromatid in G2 cells, further supporting the idea that the most DSB-induced mitotic gene conver-sion occurs via hDNA repair. Thus, z90% of Ura1/2 Leu1/1 colonies (35 of 39 from these two strains) re-sulted from palindrome segregation, giving a segrega-tion rate of z75%. The z25% nonsectored colonies might have arisen by gap repair, residual mismatch re-pair and/or loop segregation prior to plating (see dis-cussion). By ruling out gap repair for most DSB-induced
gene conversions in our system, this system can be used to address specific questions about mismatch repair in recombination intermediates.
Donor alleles are unchanged whether or not
mis-matches are repaired: Studies have demonstrated that
spontaneous and DSB-induced gene conversion is com-pletely nonreciprocal, i.e., donor loci are not altered (Nickoloff et al. 1989, J. W. Cho and J. A. Nickoloff, unpublished results). We were interested in whether donor loci are similarly unaltered when HO site con-versions are associated with at least one segregation event. To address this question, we constructed strain
Figure 2.—Sectored and nonsectored colony genotypes and phenotypes. The parental substrate is shown at the top as in Figure 1. Below are predicted products of reciprocal (or SSA) and nonreciprocal events shown as two segregation products following the first post-recombination division. Half-colony phenotypes corresponding to the URA3 and LEU2 genotypes for each segregation product are shown on the right. HO432 is assumed to be converted in at least one allele in all cases, whereas Pal may undergo full or half conversion. Only full gene conversions and simple deletions produce nonsectored colonies; all others involve sectoring of either URA3 or LEU2 or both. Half-gene conversions of Pal produce sectored Ura1/2 colonies in which both halves are either
YW14-409R, which is identical to YW14-409 except that the downstream ura3 allele carries eight phenotypically silent RFLP markers. These additional markers are well repaired (J. W. Cho and J. A. Nickoloff, unpublished results).
Markers were scored in both ura3 alleles from Ura1 and Ura2 sectors in 22 gene conversion products. Since each sector receives one DNA strand from each half of a recombination intermediate, this analysis provided information about all four participating strands of these mitotic events (two strands each from donor and recipient alleles). The unbroken X764 allele retained its parental configuration in all 22 products. Complete nonreciprocality was also seen in nonsectored intra-chromosomal gene conversion products in which 9 RFLP markers were scored (J. W. Cho and J. A. Nick-oloff, unpublished results). Thus, donor alleles
re-main unchanged whether or not all markers in hDNA are repaired.
Additional markers reduce loop segregation: The addi-tional markers in YW14-409R (relative to YW14-409) re-duced Ura1/2 sectoring from 87% to 5% among Leu1 products (Table 1). Although different HO induction times used for strains with and without silent markers preclude direct comparison of sector rates in the two types of strains, limited analysis of YW14-409R sector rates with the shorter (3 hr) induction time indicated that the silent markers reduce Ura1/2 sectoring by more than 10-fold (data not shown). Increased loop re-pair (reduced sectoring) likely reflects the corere-pair of the loop and nearby well-repaired silent markers, a well-known phenomenon in yeast (reviewed in Petes et
al. 1991). Such corepair can also explain the large
de-crease in the two measured unequal exchange classes (Ura1/2 Leu2/1 and Ura1/2 Leu1/2) in YW14-409R compared with YW14-409 and YW37-409, as corepair would shift these to the nonsectored Ura1/1 Leu1/2 class. Mismatches adjacent to an unrepaired palindromic loop mismatch are repaired efficiently: The sectored pro-ducts of strain YW14-409R can be used to examine mis-match repair of markers near an unrepaired palindro-mic loop mismatch. As mentioned above, the X764 donor allele was unchanged in both Ura1 and Ura2 sectors in all 22 products and therefore remained Ura2. Conversion and segregation patterns in the re-cipient alleles (which suffered DSBs) are summarized in Figure 3. As expected, recipient alleles of Ura1 sec-tors lost both frameshift mutations (the HO site and the palindrome). The HO site was also absent in Ura2 sectors in all 22 recipient alleles. Thus, the HO site was completely converted in all 22 products. We also ex-pected that the palindrome would be present in recipi-ent alleles in Ura2 sectors because sectoring was likely to result from loop segregation, and this was true for 21 of 22 products. In the single exception, both the loop and HO site converted, but hDNA extended to X764, which escaped repair (Figure 3, type 9).
Among these products, hybrid DNA was usually more extensive than the minimum required to pro-duce a sectored colony. This minimum is represented by the five examples of product type 1, in which only the region between the HO site and the palindrome in-sertion may have been included in hybrid DNA. The silent RFLPs are efficiently repaired in standard con-version assays (J. W. Cho and J. A. Nickoloff, unpub-lished results). Among the 17 products known to have arisen from intermediates with silent RFLPs in hDNA (types 2–9), 14 showed no evidence of silent marker segregation (types 2–6), indicating that these markers are efficiently repaired even when the nearby loop es-capes repair. These 17 products had at least 32 silent markers in hDNA, 22 of which were repaired. This 69% repair efficiency is likely an underestimate as repair that restores a marker is not detected. Short tract re-pair was evident in two products. The type 6 product had a discontinuity due to restoration of Stu463 and conversion of Bgl565. The discontinuity in the Ura2 sec-tor of the type 2 product resulted from segregation of the loop rather than repair. However, the conversion of the Nsi304 site in this product adjacent to the unre-paired loop is further evidence for short tract repair.
Of the three products in which silent markers es-caped repair (types 7–9), two showed a complete lack
of mismatch repair. Note that conversion of the HO site does not involve mismatch repair; instead, the non-homologous ends produce unpaired single-stranded tails upon invasion, and these are thought to be re-moved by Rad1/10p endonuclease (Fishman-Lobell and Haber 1992). Thus, the HO site, unlike other markers in homology, is converted by a mechanism dif-ferent from either gap or hDNA repair.
Differential involvement of markers in hDNA on op-posite sides of the DSB: Previously, we performed plas-mid 3 chromosome and intrachromosomal crosses with identical ura3 alleles as in YW14-409R except they lacked the palindrome. Among Ura1 products from these crosses, markers 59 of the DSB converted signifi-cantly more often than equidistant 39 markers ( Sweet-ser et al. 1994, J. W. Cho and J. A. Nickoloff,
unpub-lished results). We demonstrated that this asymmetry reflected a selection bias against Ura2 products that arise when tracts extending 39 of the DSB reach the X764 frameshift mutation (Weng et al. 1996). The sec-tored colonies described here were selected on the ba-sis of segregation of palindromic insertions, and 21 of 22 were of this type. Thus, selection pressure was im-posed for hDNA forming 59 from the DSB. It is there-fore striking that among sectored products, the Nsi304 marker 59 of the DSB converted at less than half the rate of the equidistant 39 marker, Bgl565 (Figure 4). Al-though a formal possibility, it is unlikely that these re-duced values reflect increased restoration repair for these markers among the sectored YW14-409R prod-ucts. Another possibility is that hDNA formation is blocked by the palindrome, and in this case, markers 59 of the palindrome would be expected to exist in hDNA at reduced levels whether the loop mismatch was re-paired or not. To test this idea, we generated a tract spectrum from 17 YW14-409R products that converted the palindrome (nonsectored Ura1/1 Leu1/1 products). As shown in Figure 4, conversion rates of 59 markers were higher than 39 markers among nonsectored prod-ucts. The tract spectrum of nonsectored YW14-409R products is essentially the same as conversion spectra ob-tained with these same alleles, but lacking the palin-drome, in both plasmid 3 chromosome and intrachro-mosomal crosses (Sweetser et al. 1994, J. W. Cho and J. A. Nickoloff, unpublished results). As this and prior
studies indicate that most DSB-induced conversion is mediated by hDNA repair, the tract spectrum of the nonsectored products would indicate that the palin-drome does not block hDNA formation. Instead, these results suggest that the 59 markers occur in hDNA less often in intermediates that produce sectored products. In contrast, the 39 markers either converted at similar or slightly higher rates in sectored recombinants com-pared with nonsectored recombinants (Figure 4). We argue below that these results indicate that conversion on opposite sides of a DSB involves distinct mismatch repair processing events.
D I S C U S S I O N
Meiotic conversion occurs at relatively high frequen-cies (a few percent or more), which allows recombi-nants to be identified without selection. This facilitates the analysis of all four strands involved in a single mei-otic recombination event. In contrast, spontaneous conversion in mitotic cells occurs about 1000-fold less often than in meiotic cells, and selection strategies are typically required to identify events that produce func-tional alleles. Selection can conceal certain types of re-combinant events. For example, selection for Ura1 products can make some unequal sister chromatid ex-change events appear as deletions. By using HO nu-clease to stimulate mitotic recombination at high fre-quencies, selection can be avoided, and information can be gained for all four strands participating in a sin-gle mitotic event. In meiosis, there are eight strands that can interact, but only rarely are more than four in-volved. The haploid mitotic system described here shows some similarity to the meiotic situation, but with different topological features. In G1 cells there is only one copy of each chromosome and only two copies of the alleles under study; thus interactions involve both available alleles. In G2 cells, most interactions are still likely to involve only two of the four copies, similar to meiosis. However, in meiotic cells all alleles are un-linked whereas in haploid mitotic cells, interacting partners are always linked in G1 cells
somal events), while in G2 cells they may be linked or unlinked (sister chromatids). In mitosis, interactions typically occur between sisters rather than homologs (Kadyk and Hartwell 1992). Our data suggest an ap-parent rate of 40% for interactions between sisters in mitosis. However, Esposito (1978) described how events initiated in G1 could give rise to products that would appear to have occurred in G2. If the events we observe are truly G2 events, the 40% value is a mini-mum estimate of sister interactions as we cannot distin-guish nonreciprocal interactions between alleles on sis-ter chromatids from intrachromosomal insis-teractions.
Most DSB-induced mitotic gene conversion is
mediat-ed by hDNA mismatch repair: DSB-induced conversion
during meiosis and MAT switching involves hDNA re-pair and our study confirms this for general mitotic DSB-induced conversion. Broken alleles are preferen-tially converted and conversion tracts are usually con-tinuous (reviewed in ref. Nickoloff and Hoekstra 1997), and these features can be explained by an end-directed, excision-based mismatch repair mechanism analogous to that mediated by MutHLS in E. coli ( Mod-rich 1991), as proposed for both MAT and meiotic
conversions in yeast (Detloff et al. 1992; Haber et al. 1993). It is not known whether spontaneous mitotic re-combination results from DSBs, other types of endoge-nous damage, or normal DNA dynamics. Similar gene conversion:crossover ratios were found for spontane-ous and DSB-induced events in two studies (Nickoloff
et al. 1986; Ray et al. 1988). These results are consistent with the idea, but do not prove, that spontaneous events are initiated by DSBs.
In the absence of other markers, we observed about 25% conversion of the palindrome. Despite our efforts to prevent cell division of recombinants prior to plat-ing, it is possible that some of the apparent palindrome conversions seen here were in fact segregation events. However, we believe that this is not the case because similar conversion rates are seen in meiosis with palin-dromes, and with point mutations in mismatch repair-deficient pms, msh, or mlh mutants (Crouse 1997), and meiotic analyses are not susceptible to this type of plat-ing artifact. Loop conversion may reflect low-level mis-match repair, either by the MLH/MSH pathway or by an unknown pathway. Msh2p-Msh6p binds to 12 and 14 base palindromic mismatches (Alani 1996). Genetic evidence is also consistent with Msh binding to palin-dromic mismatches in vivo (Manivasakam et al. 1996; Moore et al. 1988; Nag et al. 1989). It has been
sug-gested that palindromic stem-loop structures may be recognized at both the unpaired bases in the loop and at the base of the stem (Alani et al. 1995). Msh2p-Msh6p binding to single-base mismatches is reduced by ATP, but this effect is not seen for palindromic mis-matches. This differential response to ATP might ex-plain why palindromic loop mismatches are bound by Msh proteins, but not well repaired (Alani 1996).
Re-sidual loop repair might reflect an incomplete re-sponse of Msh2p-Msh6p to ATP in vivo. In addition, within the recently sequenced yeast genome are two previously unknown genes with homology to MLH and
MSH genes (Crouse 1997). These, or other unknown genes, may be responsible for residual repair of palin-dromic loops. DSBs within HO site insertions produce unpaired single-stranded tails upon invasion of a ho-mologous sequence, and Rad1/10p is thought to cleave at homology/heterology borders (Fishman-Lobell and Haber 1992). It is possible that Rad1/10p also cleaves at palindromic loop mismatches and thus con-tributes to palindrome conversion. Kirkpatrick and Petes (1997) recently showed that Rad1p is involved in
repair of nonpalindromic loop mismatches and it will be of interest to learn if Rad1p also cleaves near palin-dromic loop mismatches.
Mismatch repair in the vicinity of an unrepaired loop mismatch: The analysis of tract structures in sectored products showed that nearly 80% had hDNA extending across one or more silent markers, and more than 70% had hDNA on both sides of the DSB. These are mini-mum values because restoration repair effectively hides hDNA. Studies are in progress in mismatch repair mu-tants to more accurately identify the extent of hDNA. The amount of “hidden” hDNA may be significant be-cause the majority of silent markers were repaired, de-spite their proximity to an unrepaired loop mismatch. We did observe segregation of silent markers at a (max-imum) rate of 31%, which is about 10-fold higher (P 5 0.001) than that seen in gene conversion products of a related intrachromosomal cross lacking the palindrome (J. W. Cho and J. A. Nickoloff, unpublished results).
A marker might escape repair due to a total or par-tial lack of repair. Parpar-tial repair could reflect the termi-nation of a long repair tract prior to reaching a marker, or short tract repair acting individually on some, but not all markers. Most conversion tracts are continuous (Petes et al. 1991), consistent with excision-based, long tract repair, as in E. coli, a view that is strengthened by the identification of yeast homologs of E. coli MutL and MutS (Crouse 1997). E. coli MutH induces nicks in hemi-methylated DNA to target a strand for repair. Yeast DNA is not methylated, but for DSB-induced con-versions, the DNA ends could serve the same purpose as MutH-induced nicks (Detloff et al. 1992; Haber et
al. 1993). We found three of 22 products in which
si-lent markers escaped repair. Two showed no evidence of repair (types 7 and 8; Figure 3) and a third showed complete repair on one side of the DSB and no repair on the other (type 9). Although it is possible that the type 9 product resulted from termination of a single mismatch repair tract, we believe that it reflects inde-pendent repair processing on opposite sides of the DSB. Further evidence for this idea is discussed below.
both mitotic and meiotic cells, can result from either short tract repair or partial repair. Although short tract repair systems are known in bacteria (Lieb 1991), S. pombe
(Schar and Kohli 1993), and higher eukaryotes
(Gallinari et al. 1997; Neddermann and Jiricny 1994), previous evidence for short tract repair in yeast has been indirect, limited to observations of discontin-uous conversion tracts. In meiosis, short tract and par-tial repair can be distinguished since all strands can be followed. However, this distinction cannot be made in mitotic studies that employ selection to identify recom-binants. Using a nonselective assay, we found two prod-ucts with discontinuous tracts (Figure 3, types 2 and 6), both of which resulted from repair. In the type 2 prod-uct, repair of the Nsi304 marker did not include flank-ing Bgl205 and loop markers, each only 100 bp away. In the type 6 product adjacent markers were repaired in opposite directions. These products provide direct evi-dence of short tract repair processing in S. cerevisiae, al-beit at a low level.
Independent mismatch repair processing on oppo-sites sides of a DSB: Despite selecting for events that extended 59 of the DSB (toward the palindrome), si-lent markers 59 of the DSB were converted (or segre-gated) less often than equidistant markers 39 of the DSB. These results are opposite of previous results of related plasmid 3 chromosome and intrachromosomal crosses that lacked the palindrome (Sweetser et al. 1994; J. W. Cho and J. A. Nickoloff, unpublished re-sults). This conversion bias toward 59 markers reflects selection against longer 39 tracts, as such tracts transfer the donor X764 frameshift mutation to the recipient al-lele (Weng et al. 1996). It is unlikely that a relatively short palindrome could block branch migration in view of E. coli data showing that heterologies greater than 1 kb are incorporated into hDNA in vitro (Bianchi and Radding 1983) and in vivo (Holbeck and Smith 1992;
Lichten and Fox 1984). Although multiple mismatches
were found to inhibit pairing (Negritto et al. 1997; Worth et al. 1994), these studies involved 4- to 20-fold
more markers than the current study. We found that among products that converted the palindrome, 59 marker conversion rates were even higher (though not significantly; P 5 0.07) than rates seen in a cross lack-ing the palindrome (J. W. Cho and J. A. Nickoloff, unpublished results), thus ruling out the possibility that the palindrome prevents hDNA formation at these 59 markers. We cannot rule out the possibility that there is increased restoration repair of 59 markers among products that segregated the palindrome. However, there is no obvious mechanism that would produce such an effect. The simplest explanation for the ob-served differential marker conversion derives from the observation that poorly repaired mismatches are re-paired efficiently when near well-rere-paired mismatches (Petes et al. 1991), reflecting corepair. Such corepair indicates that poorly repaired mismatches are not
re-fractory to repair per se, but that they are unable to ini-tiate repair. For markers 59 of the DSB (on the same side as the palindrome), the reduced conversion rates in sectored products, and the normal rates in nonsec-tored products can be explained in terms of corepair. In this view, if hDNA on the 59 side of the DSB is lim-ited and includes only the palindrome, this marker is likely to escape repair and produce a sectored colony. If hDNA is more extensive, other 59 markers will be in-cluded in hDNA, and these will stimulate their own re-pair and lead to corere-pair of the palindrome, producing a nonsectored product. In contrast, conversion rates for markers 39 of the DSB were similar among sectored and nonsectored products, indicating that the pres-ence of these markers in hDNA did not stimulate core-pair of the palindrome. These results therefore suggest that conversion on opposite sides of the DSB involves independent mismatch repair tracts, and rule out mod-els involving a single mismatch repair tract.
As discussed above, our data and those of others are inconsistent with DSBs stimulating conversion in a dou-ble-strand gap. Sun et al. (1991) proposed a modified DSB repair model in which only 59 ends are degraded, producing long 39 extensions. In this model, both ends invade an unbroken, homologous duplex (donor al-lele) and prime repair synthesis (Figure 5, A–C). Reso-lution of the two HJs gives two products that each have hDNA (Figure 5D). However, Gilbertson and Stahl (1996) showed that hDNA rarely forms in unbroken al-leles, prompting the model shown in Figure 5, E–F in which only one HJ is resolved enzymatically. The reso-lution of one HJ produces an intermediate with three single-strand nicks (Figure 5E; filled triangles). Branch migration shown by the rightward arrow resolves the second junction when it reaches two of the nicks, yield-ing two duplexes, only one of which has hDNA. The nicks adjacent to hDNA (open triangles) are potential entry points for end-directed mismatch repair (Figure 5F), as suggested for meiotic conversion and MAT con-version (Detloff et al. 1992; Haber et al. 1993). Our data are consistent with hDNA forming only in broken alleles, as are data indicating that broken alleles rarely donate information to unbroken alleles (reviewed in Nickoloff and Hoekstra 1997).
conversion with a resolution level of 20–30 bp ( Sweet-ser et al. 1994), these same alleles in an
intrachromo-somal cross yielded uni- and bidirectional tracts at similar frequencies (J. W. Cho and J. A. Nickoloff, unpub-lished results). Unidirectional tracts were also predomi-nant in the meiotic study by Gilbertson and Stahl (1996), although here the level of resolution was lower ($130 bp). Thus, the model shown in Figure 5, E–F does not fully explain the available evidence.
In Figure 6 are shown two versions of a DSB repair model in which two physically separated regions of hDNA are formed on either side of the DSB; conse-quently, these regions will be processed by indepen-dent mismatch repair tracts. The first (Figure 6, A–E) is a noncrossover version of a model proposed by Gil-bertson and Stahl (see Figure 6 in Gilbertson and
Stahl 1996), and is similar to that diagrammed in
Fig-ure 5, E–F, except that mismatch repair initiates at a nick produced by enzymatic resolution of one of the two HJs (Figure 6B). After this first hDNA region is re-paired, branch migration resolves the remaining HJ (Figure 6C) producing an intermediate with a second hDNA region that is subject to an independent,
nick-directed mismatch repair tract (Figure 6D). Note that this model predicts that repair synthesis (Figure 6A) oc-curs before mismatch repair (Figure 6, B and D); how-ever, the newly synthesized DNA is completely removed and then replaced during mismatch repair. The second model (Figure 6, A9–D9) is a single-end invasion model (Belmaaza and Chartrand 1994; Nelson et al. 1996), which is essentially the same as synthesis-dependent strand annealing (Nassif et al. 1994). This model sug-gests that hDNA formed upon initial strand invasion, and/or branch migration, signals mismatch repair (Figure 6B9) before repair synthesis (Figure 6C9). This produces a strand that can anneal to the non-invading strand, yielding a second hDNA region that is subject to a second mismatch repair event (Figure 6D9). As de-scribed for Figure 5, E–F, the models in Figure 6 pre-dict that no hDNA will form in the unbroken allele.
The model shown in Figure 6, A9–D9 suggests that only one end invades a homolog; however, these same steps can be invoked in a symmetric fashion in a two-ended invasion model that also predicts independent mismatch repair tracts on opposite sides of the DSB, and yields the same products shown in Figure 6E (not shown). In these dual mismatch repair tract models, products are shown with bidirectional tracts. However, unidirectional tracts can arise if hDNA either does not form on one side of the DSB (i.e., no branch migra-tion), or by appropriate mismatch repair processing. In a cross that lacked the palindrome, 42% of intrachro-mosomal conversion tracts were bidirectional (J. W. Cho and J. A. Nickoloff, unpublished results). In
con-trast, among products in which the palindrome
segre-Figure 5.—Two versions of the DSB repair model. An un-broken homolog is shown by thick lines, repair synthesis by dashed lines, single-strand nicks by triangles, and end-di-rected mismatch repair of hDNA by “MMR” initiating at open triangles. (Left) The original DSB repair model (Szostak et al. 1983) as modified by Sun et al. (1991), in which both HJs are resolved. (Right) A model proposed by Gilbertson and Stahl (1996) to account for the lack of hDNA in unbroken alleles. In both models, mismatch repair occurs after HJs are resolved. HJs may be resolved in two senses to yield crossover products or noncrossover products; here only the latter are shown. See text for further details.
gated, there were significantly more bidirectional tracts (73%; P 5 0.01), possibly reflecting more frequent two-ended invasions. If sectored products only appear when hDNA on the 59 side of the DSB is limited and cludes only the palindrome, as argued above, such in-termediates may be stabilized by a second invasion on the 39 side of the DSB. This idea is consistent with the significantly higher level of involvement of the 39 Stu463 marker among sectored products (73%) than seen previously among conversion products from a cross lacking the palindrome (47%; P , 0.03). Conver-sion of Stu463 was slightly higher among sectored than nonsectored products of YW14-409R (Figure 4). If such effects are found to be general, it would suggest that successful recombination requires intermediates to be stabilized by a minimum length of hybrid DNA.
We thank Jennifer W. Cho and Doug Sweetser for technical
as-sistance, and Richard Kolodner, Fred Winston, Gerry Smith,
and Stephanie Ruby for helpful comments. This research was
sup-ported by grant CA55302 to J.A.N. from the National Cancer Insti-tute, National Institutes of Health.
L I T E R AT U R E C I T E D
Aguilera, A., and H. L. Klein, 1989 Yeast intrachromosomal
re-combination: long gene conversion tracts are preferentially asso-ciated with reciprocal exchange and require the RAD1 and
RAD3 gene products. Genetics 123: 683–694.
Ahn, B.-Y., and D. M. Livingston, 1986 Mitotic gene conversion
lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system. Mol. Cell. Biol. 6: 3685–3693.
Alani, E., 1996 The Saccharomyces cerevisiae Msh2 and Msh6 proteins
form a complex that specifically binds to duplex oligonucle-otides containing mismatched DNA base pairs. Mol. Cell. Biol.
16: 5604–5615.
Alani, E., N.-W. Chi, and R. D. Kolodner, 1995 The Saccharomyces
cerevisiae Msh2 protein specifically binds to duplex
oligonucle-otides containing mismatched DNA base pairs and insertions. Genes Dev. 9: 234–247.
Belmaaza, A., and P. Chartrand, 1994 One-sided invasion events
in homologous recombination at double-strand breaks. Mutat. Res. 314: 199–208.
Bianchi, M. E., and C. M. Radding, 1983 Insertions, deletions and
mismatches in heteroduplex DNA made by recA protein. Cell
35: 511–520.
Borts, R. H., and J. E. Haber, 1987 Meiotic recombination in
yeast: alteration by multiple heterozygosities. Science 237: 1459– 1465.
Borts, R. H., and J. E. Haber, 1989 Length and distribution of
meiotic gene conversion tracts and crossovers in Saccharomyces
cerevisiae. Genetics 123: 69–80.
Cao, L., E. Alani and N. Kleckner, 1990 A pathway for generation
and processing of double-strand breaks during meiotic recombi-nation in S. cerevisiae. Cell 61: 1089–1101.
Crouse, G. F., 1997 Mismatch repair systems in Saccharomyces
cerevi-siae, in DNA Damage and Repair, edited by J. A. Nickoloff and M. F. Hoekstra. Humana Press, Totowa, NJ (in press). de Massy, B., V. Rocco and A. Nicolas, 1995 The nucleotide
map-ping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae. EMBO J. 14: 4589–4598.
Detloff, P., and T. D. Petes, 1992 Measurements of excision repair
tracts formed during meiotic recombination in Saccharomyces
cere-visiae. Mol. Cell. Biol. 12: 1805–1814.
Detloff, P., M. A. White and T. D. Petes, 1992 Analysis of a gene
conversion gradient at the HIS4 locus in Saccharomyces cerevisiae. Genetics 132: 113–123.
Esposito, M. S., 1978 Evidence that spontaneous mitotic
recombi-nation occurs at the two-strand stage. Proc. Natl. Acad. Sci. USA
75: 4436–4440.
Esposito, M. S., R. M. Ramirez and C. V. Bruschi, 1994
Recombina-tors, recombinases and recombination genes of yeasts. Curr. Genet. 25: 1–11.
Fishman-Lobell, J., and J. E. Haber, 1992 Removal of
nonhomolo-gous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science 258: 480–484.
Gallinari, P., P. Neddermann and J. Jiricny, 1997 Short patch
mismatch repair in mammalian cells, in DNA Damage and Repair, edited by J. A. Nickoloff and M. F. Hoekstra. Humana Press,
Totowa, NJ (in press).
Gilbertson, L. A., and F. W. Stahl, 1996 A test of the
double-strand break repair model for meiotic recombination in
Saccha-romyces cerevisiae. Genetics 144: 27–41.
Gunn, L., J. Whelden and J. A. Nickoloff, 1995 Transfer of
episo-mal and integrated plasmids from Saccharomyces cerevisiae to
Es-cherichia coli by electroporation, pp. 55–66 in Electroporation Proto-cols for Microorganisms, edited by J. A. Nickoloff. Humana Press,
Totowa, NJ.
Haber, J. E., B. L. Ray, J. M. Kolb, and C. I. White, 1993 Rapid
ki-netics of mismatch repair of heteroduplex DNA that is formed during recombination in yeast. Proc. Natl. Acad. Sci. USA 90: 3363–3367.
Holbeck, S. L., and G. R. Smith, 1992 Chi enhances heteroduplex
DNA levels during recombination. Genetics 132: 879–891.
Holliday, R., 1964 A mechanism for gene conversion in fungi.
Genet. Res. 5: 282–304.
Judd, S. R., and T. D. Petes, 1988 Physical lengths of meiotic and
mitotic gene conversion tracts in Saccharomyces cerevisiae. Genetics
118: 401–410.
Kadyk, L. C., and L. H. Hartwell, 1992 Sister chromatids are
pre-ferred over homologs as substrates for recombinational repair in
Saccharomyces cerevisiae. Genetics 132: 387–402.
Kirkpatrick, D. T., and T. D. Petes, 1997 Repair of DNA loops
in-volves DNA-mismatch and nucleotide-excision repair proteins. Nature387: 929–931.
Lichten, M., and M. S. Fox, 1984 Evidence for inclusion of regions
of nonhomology in heteroduplex DNA products of bacterio-phage l recombination. Proc. Natl. Acad. Sci. USA 81: 7180– 7184.
Lichten, M., C. Goyon, N. P. Schultes, D. Treco, J. W. Szostak et
al., 1990 Detection of heteroduplex DNA molecules among the products of Saccharomyces cerevisiae meiosis. Proc. Natl. Acad. Sci. USA 87: 7653–7657.
Lieb, M., 1991 Spontaneous mutation at a 5-methylcytosine hotspot
is prevented by very short patch (VSP) mismatch repair. Genetics
128: 23–27.
Lovett, S. T., P. T. Drapkin, V. A. Sutera, Jr., and T. J. Gluckman-Peskind, 1993 A sister-strand exchange mechanism for
recA-independent deletion of repeated DNA sequences in Escherichia
coli. Genetics 135: 631–642.
Manivasakam, P., S. M. Rosenberg and P. J. Hastings, 1996 Poorly
repaired mismatches in heteroduplex DNA are hyper-recombi-nagenic in Saccharomyces cerevisiae. Genetics 142: 407–416.
McGill, C., B. Shafer and J. Strathern, 1989 Coconversion of
flanking sequences with homothallic switching. Cell 57: 459– 467.
Meselson, M., and C. M. Radding, 1975 A general model for
ge-netic recombination. Proc. Natl. Acad. Sci. USA 72: 358–361.
Modrich, P., 1991 Mechanisms and biological effects of mismatch
repair. Annu. Rev. Genet. 25: 229–253.
Moore, C. W., D. M. Hampsey, J. F. Ernst and F. Sherman,
1988 Differential mismatch repair can explain the dispropor-tionalities between physical distances and recombination fre-quencies of cyc1 mutations in yeast. Genetics 119: 21–34.
Nag, D. K., and T. D. Petes, 1990 Meiotic recombination between
dispersed repeated genes is associated with heteroduplex forma-tion. Mol. Cell. Biol. 10: 4420–4423.
Nag, D. K., and T. D. Petes, 1991 Seven-base-pair inverted repeats
in DNA form stable hairpins in vivo in Saccharomyces cerevisiae. Ge-netics 129: 669–673.
Nag, D. K., and T. D. Petes, 1993 Physical detection of
heterodu-plexes during meiotic recombination in the yeast Saccharomyces
Nag, D. K., M. A. White and T. D. Petes, 1989 Palindromic
se-quences in heteroduplex DNA inhibit mismatch repair in yeast. Nature 340: 318–320.
Nassif, N., J. Penny, S. Pal, W. R. Engels and G. B. Gloor,
1994 Efficient copying of nonhomologous sequences from ec-topic sites via P-element-induced gap repair. Mol. Cell. Biol. 14: 1613–1625.
Neddermann, P., and J. Jiricny, 1994 Efficient removal of uracil
from G-U mispairs by the mismatch-specific thymine DNA glyco-sylase from HeLa cells. Proc. Natl. Acad. Sci. USA 91: 1642–1646.
Negritto, M. T., X. Wu, T. Kuo, S. Chu and A. M. Bailis,
1997 Influence of DNA sequence identity on efficiency of tar-geted gene replacement. Mol. Cell. Biol. 17: 278–286.
Nelson, H. H., D. B. Sweetser and J. A. Nickoloff, 1996 Effects
of terminal nonhomology and homeology on double-strand break-induced gene conversion tract directionality. Mol. Cell. Biol. 16: 2951–2957.
Nickoloff, J. A., E. Y. C. Chen and F. Heffron, 1986 A 24-base-pair
sequence from the MAT locus stimulates intergenic recombina-tion in yeast. Proc. Natl. Acad. Sci. USA 83: 7831–7835.
Nickoloff, J. A., and M. F. Hoekstra, 1997 Double-strand break
and recombinational repair in Saccharomyces cerevisiae, in DNA
Damage and Repair, edited by J. A. Nickoloff and M. F. Hoek-stra. Humana Press, Totowa, NJ (in press).
Nickoloff, J. A., and R. J. Reynolds, 1991 Subcloning with new
ampicillin and kanamycin resistant analogs of pUC19. Biotech-niques 10: 469–472.
Nickoloff, J. A., J. D. Singer and F. Heffron, 1990 In vivo analysis
of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis. Mol. Cell. Biol. 10: 1174–1179.
Nickoloff, J. A., J. D. Singer, M. F. Hoekstra and F. Heffron,
1989 Double-strand breaks stimulate alternative mechanisms of recombination repair. J. Mol. Biol. 207: 527–541.
Orr-Weaver, T. L., J. W. Szostak and R. J. Rothstein, 1981 Yeast
transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA 78: 6354–6358.
Petes, T. D., R. E. Malone and L. S. Symington, 1991
Recombina-tion in yeast, pp. 407–521 in The Molecular and Cellular Biology of
the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and En-ergetics, edited by J. R. Broach, J. R. Pringle, and E. W. Jones.
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Priebe, S. D., J. Westmoreland, T. Nilsson-Tillgren and M. A. Resnick, 1994 Induction of recombination between
homolo-gous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair. Mol. Cell. Biol. 14: 4802–4814.
Ray, A., I. Siddiqi, A. L. Kolodkin and F. W. Stahl, 1988
Intrachro-mosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae. J. Mol. Biol. 201: 247–260.
Ray, B. L., C. I. White and J. E. Haber, 1991 Heteroduplex
forma-tion and mismatch repair of the “stuck” mutaforma-tion during mating-type switching in Saccharomyces cerevisiae. Mol. Cell. Biol. 11: 5372–5380.
Ray, F. A., E. M. Miller and J. A. Nickoloff, 1994 Efficient marker
rescue and domain replacement without fragment subcloning. Anal. Biochem. 224: 440–443.
Ronne, H., and R. Rothstein, 1988 Mitotic sectored colonies:
evi-dence of heteroduplex DNA formation during direct repeat re-combination. Proc. Natl. Acad. Sci. USA 85: 2696–2700.
Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular
Clon-ing: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
Schar, P., and J. Kohli, 1993 Marker effects of G to C transversions
on intragenic recombination and mismatch repair in
Schizosac-charomyces pombe. Genetics 133: 825–835.
Sun, H., D. Treco, N. P. Schultes and J. W. Szostak, 1989
Double-strand breaks at an initiation site for meiotic gene conversion. Nature 338: 87–90.
Sun, H., D. Treco, and J. W. Szostak, 1991 Extensive 39
-overhang-ing, single-stranded DNA associated with meiosis-specific dou-ble-strand breaks at the ARG4 recombination initiation site. Cell
64: 1155–1161.
Sweetser, D. B., H. Hough, J. F. Whelden, M. Arbuckle and J. A. Nickoloff, 1994 Fine-resolution mapping of spontaneous and
double-strand break-induced gene conversion tracts in
Saccharo-myces cerevisiae reveals reversible mitotic conversion polarity. Mol.
Cell. Biol. 14: 3863–3875.
Symington, L. S., and T. Petes, 1988 Expansions and contractions
of the genetic map relative to the physical map of yeast chromo-some III. Mol. Cell. Biol. 8: 595–604.
Szostak, J. W., T. L. Orr-Weaver, R. J. Rothstein and F. W. Stahl,
1983 The double-strand break repair model for recombina-tion. Cell 33: 25–35.
Weng, Y.-s., and J. A. Nickoloff, 1997 Nonseletive URA3
colony-color assay in yeast ade1 or ade2 mutants. Biotechniques 23: 237–242.
Weng, Y.-s., J. Whelden, L. Gunn and J. A. Nickoloff,
1996 Double-strand break-induced gene conversion: examina-tion of tract polarity and products of multiple recombinaexamina-tional repair events. Curr. Genet. 29: 335–343.
White, C. I., and J. E. Haber, 1990 Intermediates of recombination
during mating type switching in Saccharomyces cerevisiae. EMBO J.
9: 663–673.
Willis, K. K., and H. L. Klein, 1987 Intrachromosomal
recombina-tion in Saccharomyces cerevisiae: reciprocal exchange in an in-verted repeat and associated gene conversion. Genetics 117: 633–643.
Worth, L., S. Clark, M. Radman and P. Modich, 1994 Mismatch
repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs. Proc. Natl. Acad. Sci. USA 91: 3238–3241.
Wu, T. C., and M. Lichten, 1994 Meiosis-induced double-strand
break sites determined by yeast chromatin structure. Science
263: 515–518.