Incorporation of Large Heterologies Into Heteroduplex DNA During Double-Strand-Break Repair in Mouse Cells

Corresponding author: Mark D. Baker, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada., mdbaker{at}uoguelph.ca (E-mail)

Communicating editor: L. S. SYMINGTON

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

In this study, the formation and repair of large (>1 kb) insertion/deletion (I/D) heterologies during double-strand-break repair (DSBR) was investigated using a gene-targeting assay that permits efficient recovery of sequence insertion events at the haploid chromosomal immunoglobulin (Ig) µ-locus in mouse hybridoma cells. The results revealed that (i) large I/D heterologies were generated on one or both sides of the DSB and, in some cases, formed symmetrically in both homology regions; (ii) large I/D heterologies did not negatively affect the gene targeting frequency; and (iii) prior to DNA replication, the large I/D heterologies were rectified.

HOMOLOGOUS recombination represents a major pathway for the repair of chromosomal double-strand breaks (DSBs; RESNICK and MARTIN 1976 ; RUDIN and HABER 1988 ; NICKOLOFF et al. 1989 ; SARGENT et al. 1997 ; LIANG et al. 1998 ; TAKATA et al. 1998 ; LIN et al. 1999 ). In yeast, current evidence suggests that the ends of the DSB are resected by a 5'-to-3' exonuclease to produce long, 3'-ended, single-strand tails (Fig 1A; CAO et al. 1990 ; WHITE and HABER 1990 ; SUN et al. 1991 ; SUGAWARA and HABER 1992 ). In the modified version of the double-strand-break-repair (DSBR) model (SZOSTAK et al. 1983 ; SUN et al. 1991 ), one 3' end invades a homologous duplex, anneals with its complementary sequence to generate a region of asymmetric heteroduplex DNA (hDNA), and primes DNA repair synthesis, displacing a D loop. The D loop is enlarged and anneals to the second 3' end forming another region of asymmetric hDNA from which repair synthesis also initiates. This culminates in the formation of two Holliday junctions (Fig 1B). If the amount of homologous flanking DNA is sufficient, Holliday junction formation and branch migration may extend the heteroduplex in a symmetric fashion on one or both sides of the DSB (Fig 1C). Resolution of the two Holliday junctions in opposite planes results in crossover, integrating the vector into the chromosome and duplicating the region of shared homology (Fig 1D).

View larger version (22K): In this window In a new window Download PPT slide   Figure 1. DSBR model of recombination. The targeting vector and chromosomal sequences are indicated by the thick and thin lines, respectively. Regions of newly synthesized DNA primed by invading 3' vector ends are indicated by broken lines. Arrowheads denote positions where strand nicks in the joint molecule (C) would permit vector integration. Regions of asymmetric hDNA and symmetric hDNA are indicated with A and S, respectively. For further details, see text.

The formation of hDNA is important since it has the potential to accommodate sequence heterogeneities including single-base-pair mismatches or unpaired multibase insertion/deletion (I/D) loops. Current information indicates that most, if not all, meiotic and mitotic gene conversion of single-base-pair mismatches and small I/D loops results from mismatch repair (MMR) of hDNA (PETES et al. 1991 ; BOLLAG et al. 1992 ; ELLIOTT et al. 1998 ; NG and BAKER 1999 ; NICKOLOFF et al. 1999 ; ELLIOTT and JASIN 2001 ). Meiotic gene conversion of large (>1 kb) insertions and deletions occurs with an efficiency similar to that of small I/D heterologies and point mutations in yeast (PETES et al. 1991 ). Recently, a high rate of segregation of very large heterozygous insertions (2.6 and 5.6 kb) was observed during mitotic and meiotic recombination in yeast MMR mutants, suggesting that large I/D heterologies can be incorporated into hDNA and are available to cellular MMR activities (CLIKEMAN et al. 2001 ; KEARNEY et al. 2001 ).

Less is known about the behavior of large I/D heterologies in mammalian cells. In mouse cells, spontaneous conversion of a 1.5-kb insertion by intrachromosomal recombination between tandem repeats has been reported (LETSOU and LISKAY 1987 ; GODWIN and LISKAY 1994 ). The rate of conversion of the large insertion was up to two orders of magnitude lower than that of single-base-pair and small insertions at the same site. WALDMAN et al. 1999 reported a similar low rate of intrachromosomal gene conversion of a 2.4-kb insertion in mouse cells. Inserts of 114, 200, and 800 bp converted with roughly equal frequency during spontaneous intrachromosomal recombination between tandem repeats in Chinese hamster ovary (CHO) cells (SARGENT et al. 1996 ). However, since the nature of the initiating lesion was not known in these studies, a distinction between a double-strand gapped and an hDNA intermediate could not be made. Formation and efficient repair of 26-base loop mismatches during extrachromosomal recombination in CHO cells has been reported (BILL et al. 2001 ).

We report here evidence that I/D heterologies of 1.3 and 2.8 kb are incorporated into hDNA during DSB repair in mammalian cells. We utilized a gene-targeting assay in which the haploid chromosomal immunoglobulin (Ig) µ-gene in the murine hybridoma cell line, Sp6/HL (KOHLER and SHULMAN 1980 ; KOHLER et al. 1982 ), serves as the recipient for transformation with enhancer-trap insertion vectors (pTCµ_2.8 and pTC µ_2.8/1.3) bearing large deletions within the region of shared homology (Fig 2). As described previously (VALANCIUS and SMITHIES 1991 ; HASTY et al. 1992 ; DENG et al. 1993 ; LI and BAKER 2000A , LI and BAKER 2000B ; BAKER and BIRMINGHAM 2001 ), mammalian gene targeting with sequence insertion ("ends-in") vectors (THALER and STAHL 1988 ) displays several features consistent with the DSBR model of recombination (Fig 1; ORR-WEAVER et al. 1981 ; SZOSTAK et al. 1983 ; SUN et al. 1991 ), including the formation of hDNA on both sides of the initiating DSB (LI and BAKER 2000A ; BAKER and BIRMINGHAM 2001 ). In this system, it should be noted that only single crossover events result in vector integration. Previously, it was reported that repair of DSB-induced intrachromosomal homologous recombination occurs primarily through gene conversion unassociated with reciprocal exchange in mammalian cells (JOHNSON and JASIN 2000 ). Therefore, the recombinants recovered in this study may represent a fraction of the total recombination events.

View larger version (19K): In this window In a new window Download PPT slide   Figure 2. Gene targeting at the chromosomal immunoglobulin µ-locus. The structure of the recipient haploid chromosomal µ-gene in the Sp6/HL hybridoma cell line and the enhancer-trap insertion vectors, pTC µ2.8 and pTCµ2.8/1.3, is shown. The 12.6-kb vector, pTCµ2.8, contains the cloned SpeI/XbaI fragment encompassing the µ-gene switch (Sµ) and constant (Cµ) regions from the Sp6/HL hybridoma (KOHLER and SHULMAN 1980 ; KOHLER et al. 1982 ; OCHI et al. 1983 ) inserted into a pSV2neo vector backbone (SOUTHERN and BERG 1982 ). During cloning in E. coli, a 2.8-kb segment was deleted from the Sµ-region, leaving a residual, 0.4-kb Sµ-segment (OCHI et al. 1983 ; in this study, the deletion is denoted M1* to distinguish it from the wild-type chromosomal sequence M1). The Sµ-deletion does not affect the ability of this fragment to encode a functional µ-chain (OCHI et al. 1983 ). The 11.3-kb vector, pTCµ2.8/1.3, was derived from pTCµ2.8 by deleting the 1.3-kb DraIII/EcoRV segment (denoted M2* to distinguish it from the wild-type chromosomal sequence, M2) from the Cµ-region. In each vector backbone, the 372-bp NsiI/NdeI fragment encompassing the SV40 early region enhancer was deleted. As described previously (BAUTISTA and SHULMAN 1993 ; NG and BAKER 1998 ; NG and BAKER 1999 ), the enhancer-trap feature enriches for gene-targeting events at the chromosomal Ig µ-locus. In both vectors, cutting at the unique XbaI site creates the DSB. Transfection of vector DNA (8.7 pmol) into 2 x 107 recipient Sp6/HL hybridoma cells was performed by electroporation according to conditions described previously (BAKER et al. 1988 ). According to trypan blue exclusion, hybridoma cell survival averages 50%. Following electroporation, the surviving hybridoma cells were distributed at a low cell density to the individual wells of 96-well tissue culture plates and placed under G418 selection as described earlier (NG and BAKER 1999 ; LI and BAKER 2000A , LI and BAKER 2000B ). These procedures ensure, with high likelihood, that individual transformants represent the progeny of single G418R cells deposited in the culture wells (NG and BAKER 1999 ; LI and BAKER 2000A , LI and BAKER 2000B ). Genomic DNA was prepared from each G418R transformant by the method of GROSS-BELLARD et al. 1973 and subjected to Southern analysis to identify correctly targeted clones and to assign chromosomal I/D markers in the recombinant µ-locus. For the recipient Sp6/HL µ-gene, the fragment sizes generated following digestion with EcoRI and PacI/PaeR7I are presented. Probe F is an 870-bp XbaI/BamHI Cµ-region fragment. VHTNP, TNP-specific µ-heavy-chain variable region; Sµ, µ-gene switch region; Cµ, µ-gene constant region; neo, neomycin phosphotransferase gene; E, EcoRI; Pa, PaeR7I; Pc, PacI; Sp, SpeI; Xb, XbaI. The figure is not drawn to scale.

The 12.6-kb vector, pTCµ_2.8, bears a 2.8-kb deletion (denoted M1* to distinguish it from the wild-type M1 chromosomal sequence) on the 5' side of the DSB (Fig 2). Targeted integration of a single copy of the vector into the chromosomal µ-locus generates a tandem duplication of the shared Cµ-region of homology. As shown in Fig 3A, four possible marker patterns are distinguishable in Southern analysis by diagnostic EcoRI fragments. Four separate electroporations yielded 45 independent, single-copy targeted recombinants (Table 1). The marker pattern in each recombinant is presented in Fig 3B. As an example of the Southern analysis, Fig 3C presents some representative G418^R transformants.

View larger version (112K): In this window In a new window Download PPT slide   Figure 3. Analysis of µ-gene structures in recombinants generated by targeted integration of the vector, pTC µ2.8. (A) Targeted integration of a single copy of the vector, pTCµ2.8, into the Sp6/HL chromosomal µ-gene generates a duplication of the Cµ-region of homology separated by the integrated vector sequences. As explained in the text, combinations of the M1 chromosomal and M1* vector markers in the 5' and 3' Cµ-regions yields four possible targeted vector integration patterns in the recombinants (designated i–iv). For each recombinant µ-gene structure, the diagnostic fragment sizes generated following digestion with EcoRI and hybridization with Cµ-probe F (Fig 2) are presented. (B) Analysis of recombinant µ-gene marker patterns in recombinants generated by targeted integration of pTCµ2.8. For clarity, only the genetic markers are shown. (C) Southern analysis of the µ-gene structure in representative G418R transformants. Hybridoma genomic DNA was digested with EcoRI, electrophoresed through a 0.7% agarose gel, blotted to nitrocellulose, and hybridized with 32P-labeled Cµ-specific probe F (Fig 2). The hybridoma cell line, igm10, is an Sp6-derived mutant that has lost the chromosomal Ig µ-gene (KOHLER and SHULMAN 1980 ) and was included as a control for the specificity of probe F. The sizes of relevant DNA marker bands are shown on the left of the blot while the sizes of the bands of interest are shown on the right. Abbreviations are the same as in Fig 2. The figures are not drawn to scale.

The majority of the recombinants (33/45) contained a recombinant µ-locus that bears the chromosomal (M1) marker in the 5' Cµ-region and the vector-borne (M1*) marker in the 3' Cµ-region [pattern in Fig 3A(i) and recombinants 9/1, 41/1, 49/1, and 52/1 in Fig 3C]. As can be deduced from Fig 2, this class is explained most simply by a single reciprocal crossover event on the DSB proximal side of the heterology. Five recombinants (7/1, 10/1, 12/1, 27/1, and 33/1) had chromosomal (M1) markers in both Cµ-regions of homology [pattern in Fig 3A(ii) and recombinant 27/1 in Fig 3C]. These recombinants are consistent with a gene conversion event toward the chromosomal sequence, which might arise as a consequence of enlargement of the initial DSB to form a double-strand gap (DSG) that, if large enough to remove the vector-borne M1* marker, could be repaired by DNA synthesis as shown in Fig 1. Alternatively, the M1* marker may remain and, following strand invasion or Holliday junction branch migration, be incorporated into hDNA of the general structure depicted in Fig 1D. In this case, the conversion tracts in the 5' and 3' Cµ-regions in the recombinants would be consistent with MMR of hDNA, such as observed previously for simple mismatches (NG and BAKER 1999 ; NICKOLOFF et al. 1999 ).

The marker patterns in the remaining seven recombinants provide strong support for incorporation of the large heterology into an hDNA intermediate. Recombinants 24/1 and 42/1 contained the vector-borne (M1*) marker in both regions of homology [pattern in Fig 3A(iv) and recombinant 42/1 in Fig 3C], signifying events in which genetic information was transferred from the linearized vector to the unbroken chromosome. This situation is most consistent with MMR of the hDNA intermediate shown in Fig 1D in the direction of the M1* marker. In the five remaining recombinants (2/1, 16/1, 20/1, 36/1, and 38/1), the M1* and M1 markers reside in the 5' and the 3' Cµ-regions, respectively [pattern in Fig 3A(iii) and recombinants 2/1 and 16/1 in Fig 3C]. These recombinants could have been generated by a crossover event initiating in the 1.3 kb of homology on the 5' side of marker M1*. However, this situation would require that the vector recircularize and initiate homologous recombination within this small region. Although we cannot exclude this mechanism, we believe the results are more consistent with initiation of homologous recombination at the site of the initial DSB at XbaI, with the final products being generated by MMR of the hDNA intermediate presented in Fig 1D.

Of the remaining 714 G418^R transformants, three bore EcoRI Cµ-fragments of 16.2, 12.6, and 8.9 kb. As indicated above, the unit length pTCµ_2.8 vector is 12.6 kb. Therefore, these fragment sizes are diagnostic of recombinants bearing a Cµ-region triplication resulting from the targeted integration of two copies of the transfer vector in tandem orientation. The final 711 transformants lack the EcoRI fragments indicative of targeted vector integration (for example, in Fig 3C, transformants 34/1, 40/1, and 48/1). The majority of these (703/711) retained the endogenous, 12.5-kb EcoRI µ-gene fragment with evidence in many of one or more variable-sized fragments, likely representing cases of random integration of the targeting vector into the hybridoma genome. The 8 remaining transformants contained Cµ-hybridizing fragments of unexpected size. Hybridoma cell lines with unexpected Cµ-region fragments have been observed in previous gene-targeting studies (BAKER and READ 1993 ; NG and BAKER 1999 ; LI and BAKER 2000B ; BAKER and BIRMINGHAM 2001 ). Although they have not been fully characterized, these may represent cases where one arm of the vector has been degraded, forcing the cells to undergo one-sided recombination with the target locus or perhaps illegitimate recombination such as has been reported previously (KANG and SHULMAN 1991 ; BERINSTEIN et al. 1992 ; BAKER and READ 1993 ).

The 11.3-kb vector, pTCµ_2.8/1.3, differs from the endogenous µ-locus by a large deletion on each side of the DSB. The vector-borne markers are denoted M1* and M2* to distinguish them from the corresponding wild-type µ-locus sequences, M1 and M2, respectively (Fig 2). As depicted in Fig 4A, four possible marker combinations can be generated in each of the 5' and 3' Cµ-regions. For simplicity, they are illustrated separately; however, each pattern in the left column can potentially be found with one in the right, yielding a total of 16 possible Cµ-region marker patterns. Southern blot analysis identified 73 transformants from two separate electroporations of pTCµ_2.8/1.3 that bore diagnostic EcoRI Cµ-fragments expected for integration of a single copy of the vector into the target locus (Table 1). The marker pattern in each targeted recombinant is presented in Fig 4B. The criteria used in interpreting the marker patterns in each recombinant were the same as those utilized in the preceding section. On the basis of the mechanism(s), the 16 possible marker patterns are divided into five classes. As was seen with pTCµ_2.8, the vast majority of recombinants (55/73) had a marker pattern that was most consistent with vector integration via a single crossover at or near the DSB (class I). Class II is represented by five recombinants (49/2, 52/2, 74/2, 88/2, and 95/3). In cell lines 52/2, 74/2, 88/2, and 95/3, vector-borne markers (M1* and/or M2*) are present at equivalent positions in both regions of homology. Cell line 49/2 contains the M1* and M2 markers in the 5' Cµ-region, while in the 3' Cµ-region, the M1 and M2* markers are present. The marker patterns in these recombinants are most consistent with MMR of an hDNA tract that encompassed the 2.8-kb and/or the 1.3-kb heterologies in both participating regions of homology. Seven class III recombinants (2/2, 10/2, 24/2, 32/2, 47/2, 55/3, and 68/3) display a pattern in which, on one side of the DSB, equivalent positions in both regions of homology contain either the chromosomal M1 or M2 marker, while, in equivalent positions on the opposite side of the DSB, both the vector-borne and chromosomal markers are present. While consistent with the possibility of MMR of the hDNA intermediate shown in Fig 1D, chromosomal sequences residing on only one side of the DSB might also arise by asymmetric DSG formation and repair. The final class III recombinant (109/3) contains all chromosomal markers in both the 5' and 3' regions of homology. This marker pattern could have arisen by MMR of hDNA or by repair of a DSG that encompassed the vector-borne M1* and M2* markers. Class IV is represented by the two recombinants, 45/3 and 71/3. In these cell lines, the M1 and M2 markers reside in the 5' Cµ-region, while the M1* and M2* markers reside in the 3' Cµ-region. Similarly, in class V recombinants, 31/2, 67/2, and 108/2, the M1* and M2* markers reside in the 5' Cµ-region, while the M1 and M2 markers reside in the 3' Cµ-region. As with the five recombinants generated by transfer of pTCµ_2.8 described above (2/1, 16/1, 20/1, 36/1, and 38/1), we cannot exclude the possibility that the class IV and V recombinants might have arisen as a consequence of crossover within the 1.0 kb of homology on the 3' side of the vector-borne M2* marker (class IV) or within the 1.3 kb of homology on the 5' side of the vector-borne M1* marker (class V).

View larger version (49K): In this window In a new window Download PPT slide   Figure 4. Analysis of µ-gene structures in recombinants generated by the targeted integration of the vector, pTC µ2.8/1.3. (A) Targeted integration of a single copy of the vector pTCµ2.8/1.3 into the Sp6/HL chromosomal µ-gene generates a duplication of the Cµ-region of homology separated by the integrated vector sequences. As described in the text, the various combinations of chromosomal (M1 and M2) and vector-borne (M1* and M2*) markers in the 5' and 3' Cµ-regions yield 16 possible targeted vector integration patterns. To unambiguously distinguish the diagnostic 12.1-kb EcoRI fragment in transformants bearing the M1* and M2* markers in the 5' Cµ-region from the endogenous 12.5-kb EcoRI fragment that would be expected in transformants bearing a randomly integrated vector, genomic DNA from applicable cell lines was digested with the combination PacI/PaeR7I, which cuts outside the transferred vector, and subjected to Southern analysis with Cµ-probe F (Fig 2). Random transformants contain the endogenous 14.8-kb PacI/PaeR7I Cµ-region fragment (Fig 2) together with a second, Cµ-hybridizing fragment of a variable size from the ectopic vector integration site, whereas correctly targeted cell lines contain both Cµ-regions linked on a PacI/PaeR7I fragment whose size depends on the genetic marker combination present. (B) Analysis of recombinant µ-gene marker patterns in targeted recombinants generated by targeted integration of pTCµ2.8/1.3. For clarity, only the genetic markers are shown. The marker patterns are grouped into classes I–V on the basis of their likely mechanism(s) of formation, as described in the text. Abbreviations are the same as in Fig 2. The figures are not drawn to scale.

Of the remaining 227 G418^R transformants analyzed, 6 contained EcoRI fragments of 14.9, 11.3, and 8.9 kb and 1 contained EcoRI fragments of 14.9, 11.3, and 7.6 kb. The 11.3-kb band is the size of the unit length pTCµ_2.8/1.3 vector, and its presence with the 14.9- and 8.9-kb EcoRI fragments or the 14.9- and 7.6-kb EcoRI fragments is consistent with a Cµ-region triplication resulting from targeted integration of two tandem vector copies. The balance of the 220 G418^R transformants did not contain the EcoRI fragments indicative of targeted vector integration.

Recombinants bearing vector-borne markers at equivalent positions in both participating regions of homology (recombinants 24/1, 42/1, 52/2, 74/2, 88/2, and 95/3) are consistent with the possibility of the I/D heterology being incorporated in symmetric hDNA, with subsequent repair of the mismatches occurring toward the vector-borne sequences. This would occur if 5'-to-3' resection of the DSB did not proceed past the heterology and, following strand invasion by the 3' end (Fig 1B), the Holliday junction was translated through the heterology by branch migration (Fig 1C). In support of this, we have previously demonstrated the occurrence of symmetric hDNA during targeted vector integration in mammalian cells, as evidenced by sectoring of small palindromic markers at equivalent positions in both participating regions of homology at the recombinant µ-locus (BAKER and BIRMINGHAM 2001 ). Alternatively, extensive resection of the broken DNA ends might occur past the heterology. Strand invasion by a 3' end would then incorporate the heterology in a region of asymmetric hDNA (Fig 1B) that is repaired in favor of the vector-borne marker. DNA repair synthesis directed from the second 3' end fills in the gap using the chromosomal strand in the displaced D loop as a template (Fig 1B). Reverse branch migration through the heterology would generate a region of symmetric hDNA, as in the first mechanism. Other mechanisms could also produce this marker pattern (ALLERS and LICHTEN 2001 ).

The inhibitory influence of the MMR system on homologous recombination between diverged substrates has been well documented in both yeast and mammalian cells (reviewed in EVANS and ALANI 2000 ; HARFE and JINKS-ROBERTSON 2000 ). Even a single-base-pair mismatch within a region of otherwise perfect identity is sufficient to reduce spontaneous mitotic intrachromosomal recombination rates up to fivefold (DATTA et al. 1997 ; CHEN and JINKS-ROBERTSON 1999 ; LUKACSOVICH and WALDMAN 1999 ). The absolute frequencies of gene targeting obtained in this study (1 x 10^-6 recombinants/cell and 3 x 10^-6 recombinants/cell for the vectors, pTCµ_2.8 and pTCµ_2.8/1.3, respectively; Table 1) are similar to those reported previously (1.7 x 10^-6 recombinants/cell) for enhancer-trap vectors in which the Cµ-region was either wild type (NG and BAKER 1998 ) or marked with simple restriction enzyme site polymorphisms (NG and BAKER 1999 ). Thus, the results suggest that the presence of a large heterology on one or both sides of the DSB did not negatively impact the efficiency of the mammalian gene-targeting reaction. The reason for the discrepant results might be due to the large amount of perfect homology (>1.6 kb) between the DSB and the beginning of the I/D heterologies. This might make it less likely for hDNA to have the opportunity of forming and explain why the marker pattern in the majority of recombinants (88/118) is consistent with vector integration via a crossover on the DSB proximal side of the heterologies. The length of uninterrupted homology adjacent to the DSB in our study is expected to be sufficient for efficient initiation of recombination (WALDMAN and LISKAY 1988 ; PRIEBE et al. 1994 ; ELLIOTT et al. 1998 ). Thereafter, strand transfer might be able to propagate through the heterologies, such as has been suggested previously (WALDMAN and LISKAY 1988 ). This is also suggested by in vitro studies that have shown that the ability of Escherichia coli RecA protein and its eukaryotic homolog Rad51 to bypass heterologies during strand transfer is limited to insertions (or deletions) of <250 and <9 bp, respectively (IYPE et al. 1994 ; MOREL et al. 1994 ; HOLMES et al. 2001 ). However, in the presence of single-strand binding protein, the E. coli RuvAB complex can act on RecA strand exchange intermediates to mediate bypass of large (>1 kb) heterologous insertions (IYPE et al. 1994 ; PARSONS et al. 1995 ; ADAMS and WEST 1996 ). Recent studies have identified protein complexes in mammalian cells that exhibit branch migration and Holliday junction resolution activities similar to RuvABC (CHEN et al. 2001 ; CONSTANTINOU et al. 2001 ).

Previous reports showed that spontaneous intrachromosomal gene conversion of a 1.5-kb insertion was up to two orders of magnitude lower than that of single-base-pair and small insertions at the same site in mouse cells (LETSOU and LISKAY 1987 ; GODWIN and LISKAY 1994 ). The authors postulated that the large heterologies interfered with the formation of the conversion intermediate. In this study, the apparent efficiency with which large heterologies are encompassed within hDNA during recombination between a transferred plasmid and chromosome might suggest that topological constraints interfere with this process during intrachromosomal recombination between closely linked substrates. Alternatively, the disparity in the results might reflect differences in the pathways of spontaneous intrachromosomal gene conversion and DSB-induced homologous recombination between a transfected plasmid and a chromosome.

Inclusion of large I/D heterologies in a heteroduplex region is expected to generate large, single-strand loop mismatches. As described previously in studies utilizing small palindrome genetic markers (LI and BAKER 2000A , LI and BAKER 2000B ; BAKER and BIRMINGHAM 2001 ), the recombinant isolation procedures utilized here would have permitted the detection of any unrepaired mismatch as sectored (mixed) colonies. Thus, the absence of sectoring demonstrates that the mismatches were efficiently repaired in vivo prior to DNA replication and division of the single cell undergoing recombination. Previously, repair of large loop mismatches in mammalian cells has been suggested, but from the results of injecting preformed heteroduplexes (AYARES et al. 1987 ; WEISS and WILSON 1987 , WEISS and WILSON 1989 ) as well as from studies examining recombination between extrachromosomal plasmids (BILL et al. 2001 ).

Current evidence suggests that multiple, overlapping loop repair pathways are active in yeast cells. Small loops [<15 nucleotides (nt)] are efficiently rectified by the general MMR pathway (KRAMER et al. 1989 ; TRAN et al. 1996 ; SIA et al. 1997 ; LUHR et al. 1998 ). Larger (16- to 283-nt) loops are repaired primarily via an MMR-independent mechanism (TRAN et al. 1996 ; SIA et al. 1997 ; CORRETTE-BENNETT et al. 1999 ; HARFE and JINKS-ROBERTSON 1999 ; CORRETTE-BENNETT et al. 2001 ). In contrast, others have reported a requirement for MMR proteins in the repair of 26- and 94-nt loops (KIRKPATRICK and PETES 1997 ; HARFE and JINKS-ROBERTSON 1999 ). Both MMR-dependent and -independent repair of very large loops (>2 kb) have been reported in yeast (CLIKEMAN et al. 2001 ; KEARNEY et al. 2001 ). Repair of both small and large loops also requires the nucleotide excision repair Rad1/Rad10 junction-specific endonuclease complex (KIRKPATRICK and PETES 1997 ; KIRKPATRICK 1999 ; NICHOLSON et al. 2000 ; KEARNEY et al. 2001 ). Evidence for multiple loop repair pathways has also been suggested from studies performed in mammalian cells (UMAR et al. 1994 ; LITTMAN et al. 1999 ; BILL et al. 2001 ). The Ercc1/Xpf complex, considered to be the mammalian equivalent to the yeast Rad1/Rad10 complex (SIJBERS et al. 1996 ), may also play a role in the repair of loop mismatches, although, in one study, loop repair was independent of this complex (LITTMAN et al. 1999 ).

	ACKNOWLEDGMENTS

We thank Leah Read for providing excellent technical assistance and the members of our laboratory for helpful comments during the course of this work. This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) to M.D.B. and a CIHR studentship to S.J.R.

Manuscript received February 5, 2002; Accepted for publication July 1, 2002.

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