Evidence for Biased Holliday Junction Cleavage and Mismatch Repair Directed by Junction Cuts during Double-Strand-Break Repair in Mammalian Cells

Abstract

In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.In the yeast Saccharomyces cerevisiae, transferred DNA undergoes homologous recombination with cognate chromosomal sequences (gene targeting) according to the doublestrand-break repair (DSBR) model of recombination (34,37,44). Features of meiotic recombination in S. cerevisiae are also consistent with repair of chromosomal double-strand breaks (DSBs) by this model (15,33,35,39,40,44). According to the canonical DSBR model (34, 37, 44) and its later revision (42), as illustrated in Fig. 1 for a typical gene targeting reaction, recombination is initiated by a DSB in the vector-borne region of homology to the chromosome. The DSB undergoes 5Ј33Ј resection (Fig. 1A) resulting in the formation of two 3Ј-ending single strands which invade cognate chromosomal sequences (Fig. 1B). The invading 3Ј ends prime DNA synthesis, finally generating two Holliday junctions (Fig. 1C). Opposite-sense cleavage of the Holliday junctions in the joint molecule (Fig. 1D) results in crossover, integrating the vector into the chromosome and duplicating the region of shared homology ( Fig. 1E and F).Our laboratory has been investigating mechanisms of homologous recombination in mammalian somatic cells using a gene targeting assay as one approach. By examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair (MMR) when encompassed within heteroduplex DNA (hDNA) formed in vivo during homologous recombination, we have shown that (i) hDNA is formed on each side of the vector-borne DSB and (ii) palindrome markers in hDNA formed in each homology region reside in a tr...

Analysis of One-Sided Marker Segregation Patterns Resulting From Mammalian Gene Targeting

“…The enhancer-trap nature of the targeting vectors permitted independent G418 R transformants to be isolated by limited dilution cloning as detailed elsewhere (Ng and Baker 1999a;Baker and Birmingham 2001). Genomic DNA was prepared from G418 R transformants by SDS-proteinase K digestion (Gross-Bellard et al 1973).…”

“…The Cm region of homology was inserted into a SalI site that replaced the unique EcoRI site in pSV2neo. The vector-borne Cm region was engineered so that unique BamHI and ApaI sites were replaced with palindrome insertions as described (Baker and Birmingham 2001). The palindrome contains a novel NotI site (indicated by boldface type) (59-GTACTGTATGTGCGGCCGCACATACAGTAC-39) to facilitate discrimination between sequences of vector and chromosomal origin (Baker and Birmingham 2001).…”

“…The enhancer-trap gene-targeting vectors differ solely in the amount of homology that they share with the Sp6/HL chromosomal Cm region ( Figure 5E) and are designated according to how much homology resides on the two sides of the DSB: pCmpal Long/Long contains a 4531-bp MscI/ Acc65I fragment ( Figure 5A), pCmpal Short/Short contains a 2587-bp XbaI/FspAI fragment ( Figure 5B), pCmpal Short/Long contains a 4303-bp XbaI/XbaI fragment ( Figure 5C), and pCmpal Long/Short contains a 3711-bp MscI/FspAI fragment ( Figure 5D). Plasmid propagation, purification, and manipulation were performed as described (Sambrook et al 1989;Baker and Birmingham 2001).…”

“…However, this expectation is not fulfilled in studies of meiotic recombination in yeast (Porter et al 1993;Gilbertson and Stahl 1996;Foss et al 1999;Merker et al 2003;Jessop et al 2005) or mammalian gene targeting (Baker and Birmingham 2001;Birmingham et al 2004). Instead, a considerable fraction of recombination events are one sided; that is, an FC (6:2) or HC (5:3) event on one side of the DSB is usually found in conjunction with normal 4:4 segregation on the other side of the DSB.…”

“…The crossover products in Figure 1, F and G, differ with respect to the arrangement of asymmetric hDNA (A) and gene conversion (GC c ) tracts about the DSB in the two homology regions. Although the canonical DSBR model depicts formation of both crossover products, a bias yielding products predominantly of the 2,4 resolution type ( Figure 1F) is observed (Foss et al 1999;Baker and Birmingham 2001;Merker et al 2003;Jessop et al 2005). To account for the bias in recovery of type 2,4 resolution products, Foss et al (1999) propose that each Holliday junction bears a structural asymmetry resulting from new DNA, or its synthesis, that biases the pair of like strands that are cleaved at each junction: with respect to Figure 1, crossed strands in the Holliday junction to the right of the DSB and noncrossed strands in the Holliday junction to the left of the DSB.…”