The Saccharomyces cerevisiae Msh2 Mismatch Repair Protein Localizes to Recombination Intermediates In Vivo  Elizabeth Evans, Neal Sugawara, James E Haber, Eric Alani  Molecular Cell  Volume 5, Issue 5, Pages 789-799 (May 2000) DOI: 10.1016/S1097-2765(00)80319-6

Figure 1 Diagram of Recombination Substrates Used in This Study and a Proposed Model for the Removal of Nonhomologous DNA Ends during Double-Strand Break Repair (A) Schematic diagram of plasmids. pFP122-H contains inverted repeats of E. coli lacZ sequences (black bars) that can undergo homologous recombination. One copy (the recipient) contains a HO endonuclease cleavage site (HOcs), while the donor has a single nucleotide change that prevents HO cleavage. In pFP120-TN, an internal 918 bp segment of the lacZ donor has been deleted (dashed line), resulting in a block of nonhomology in the recipient sequence flanking the HO cut site (hatched bars). In pFP125-N, the donor sequence has been removed (dashed line). Sites A–E represent the region amplified by primer pairs used in chromatin IP. Site C spans a unique junction in the donor copy of lacZ in pFP120-TN due to the deletion. (B) Model for removal of nonhomologous ends during double-strand break repair (Sugawara et al. 1997). (I) A DSB is formed in nonhomologous sequences embedded within a homologous region. (II) DSB ends are processed to form single-stranded tails that engage in a homology search. (III) Pairing of homologous sequences leads to extrusion of an unpaired tail. Msh2p-Msh3p binds and stabilizes the junction between homologous and nonhomologous DNA, allowing cleavage by Rad1p-Rad10p. (IV) Repair synthesis leads to completion of gene conversion with or without crossing over. Molecular Cell 2000 5, 789-799DOI: (10.1016/S1097-2765(00)80319-6)

Figure 2 Msh2p Specifically Cross-Links to Plasmids Induced for DSBR by HO Endonuclease Cleavage EAY668 was transformed with pFP120-TN (EAY642), pFP125-N (EAY643), and pFP122-H (EAY673). The resulting strains were grown in selective media, transferred to YP + 2% lactate, and grown to mid log phase at 30°C. Formation of the double-strand break by HO endonuclease was induced by the addition of 2% galactose. At the indicated times, cell aliquots were treated with formaldehyde and subjected to chromatin IP with 12CA5 antibody. Input extract (no IP) was processed in parallel. Following reversal of the cross-links, purified DNA was used as the template for PCR with the indicated primer pair (see Experimental Procedures). Products were visualized by agarose gel electrophoresis, and reverse images of ethidium bromide stained gels are shown. (A) (Left) Products of 28 cycles of PCRs with primer pair A (recipient) and B (vector) using DNA from a time course of EAY642 (pFP120-TN) with IP for Msh2p (upper) or with no IP (lower). (Right) The top graph displays the relative increase in PCR product formed at site A (open squares) and site E (open circles) following chromatin IP of cells induced with galactose, or at site A for cells mock induced with glucose (closed circles). The bottom graph displays the results of PCR at site B (open squares) and site D (open circles) from chromatin IP after galactose induction. (B) (Left) Products of 28 cycles of PCR with primer pairs A and B using DNA derived from the time course of EAY643 (pFP125-N) with IP for Msh2p (upper) or with no IP (lower). (Right) The top graph displays the relative increase in PCR product formed at site A after chromatin IP (squares). The bottom graph displays the relative increase in PCR product at site B (squares). (C) (Left) Products of 27 cycles of PCR with primer pair A using DNA from a time course of EAY673 (pFP122-H) with IP for Msh2p (top) or with no IP (bottom). (Right) The graph displays the relative increase in PCR product formed after chromatin IP of cells induced with galactose at site A (squares). Molecular Cell 2000 5, 789-799DOI: (10.1016/S1097-2765(00)80319-6)

Figure 3 Msh2p Interacts with Donor Sequences on pFP120-TN DNA derived from a EAY642 time course was analyzed as described in Figure 2A, using PCR primer pair A (recipient, open squares) and primer pair C (donor, closed circles). Molecular Cell 2000 5, 789-799DOI: (10.1016/S1097-2765(00)80319-6)

Figure 4 Msh2p Cross-Linking Near the DSB Site Depends on Msh3p and Rad50p, but Not Msh6p or Rad1p Wild type (EAY642; see Figure 2B), and msh3Δ (EAY679), msh6Δ (EAY678), rad1Δ (EAY675), and rad50Δ (EAY676) deletion derivatives carrying pFP125-N were induced for DSB formation and analyzed by chromatin IP for Msh2p as described in Figure 2B using PCR primer pair A (recipient). Experiments were each repeated two to three times; representative data are shown. Molecular Cell 2000 5, 789-799DOI: (10.1016/S1097-2765(00)80319-6)

Figure 5 Msh2p Localizes to DSBR Intermediates prior to the Requirement for Rad52p Experimental and PCR conditions were as described in Figure 2A (pFP120-TN) and 2C (pFP122-H). Purified DNA from no IP (input) and IP samples was subjected to PCR with primer pairs A (recipient), B (vector), and C (donor) as indicated. (A) Chromatin IP time course of a rad52Δ strain carrying pFP120-TN (EAY672). In the graph, open squares represent relative PCR amplification at site A (recipient) and closed circles represent site C (donor). (B) Chromatin IP time course of a rad52Δ strain carrying pFP122-H (EAY674). A short time course of a wild-type strain carrying pFP122-H (EAY673) is also shown (also see Figure 2C). The upper graph depicts the experiment shown to the right with PCR amplification at recipient site A for rad52Δ (open squares), and wild type (closed circles). The lower graph represents the same analysis of an independent time course. Molecular Cell 2000 5, 789-799DOI: (10.1016/S1097-2765(00)80319-6)

Figure 6 Two Steps in Recombination in which the Msh2p-Msh3p Complex May Interact with Recombination Intermediates (A) Msh2p-Msh3p loads onto DSB sites at recessed ends (1) and/or plays an active role in scanning hDNA, interacting with mispairs formed during pairing of homeologous sequences (2), leading to their rejection from heteroduplex. (B) Msh2p-Msh3p binds at the junction of homologous and nonhomologous DNA allowing for cleavage of unpaired tails by Rad1p-Rad10p (3). Molecular Cell 2000 5, 789-799DOI: (10.1016/S1097-2765(00)80319-6)