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. 2010 May 27;6(5):e1000973. doi: 10.1371/journal.pgen.1000973

Figure 1. Experimental systems of break-induced replication (BIR) and gene conversion (GC).

Figure 1

(A) In the experimental system to study BIR, an HPHMX marked HO cut site (gray bar) is integrated into the CAN1 gene on Ch V, deleting the 3′ end portion of the gene, the remaining sequences are represented as CA. The AN1 donor sharing 1,157 bp homology with CAN1 is integrated into Ch XI. PCR with primers P1 and P2 monitors the initiation of new DNA synthesis while PCR with primers P1 and P4 detects synthesis past the AN1 sequences, specifc to the donor sequences on Ch XI. Southern blot analysis of AvaI-digested (marked by “A”) DNA probed with CAN1 sequences monitors extension of the BIR fork. Completion of BIR is monitored by Pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis using the MCH2 sequences that are duplicated when the entire donor chromosome arm is copied. (B) In the experimental system to study ectopic GC. A galactose inducible HO endonuclease generates a DSB within the CAN1 locus (disrupted by URA3 creating a 376 bp gap) on Ch V. An additional 2,404 bp of homologous sequences to the gene conversion donor sequences found on Ch XI are distal to the cut site and are denoted as “1.” PCR with primers P1 and P2 monitors both the starting strain and repair into the CAN1 sequences. PCR with primers P1 and P3 monitors repair by GC in which the distal end of the break is retained.