RAD51-Dependent Break-Induced Replication Differs in Kinetics and Checkpoint Responses from RAD51-Mediated Gene Conversion

Abstract

Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time. However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms. Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome. Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G 2 /M DNA damage checkpoint. RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process. RAD51-dependent BIR occurs efficiently in G 2 -arrested cells. Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.Eukaryotic cells have evolved several mechanisms to repair broken chromosomes (reviewed in references 28 and 34). In Saccharomyces cerevisiae, gene conversion (GC) is the dominant pathway for double-strand break (DSB) repair. GC requires both ends of a DSB to share homology with sequences located on a sister chromatid, on a homologous chromosome, or in some ectopic location. This homology is used as a template for DSB repair, resulting in a limited patch of new DNA synthesis. In mitotic cells of budding yeast, GC generally occurs without an exchange of flanking chromosome arms, although conversions are accompanied by crossing over 5 to 10% of the time (10, 12). GC depends on the Rad51 strand exchange protein, as well as the recombination proteins Rad52, Rad54, Rad55, and Rad57 (11,34).In diploids, a DSB is repaired by GC Ͼ98% of the time, making other DSB repair mechanisms difficult to study. When GC is eliminated, by deleting the RAD51 gene, a DSB in the middle of a chromosome can be repaired by RAD52-dependent break-induced replication (BIR), in which sequences centromere-proximal to the DSB invade the unbroken homologous chromosome, establish a replication fork, and copy the template chromosome to the chromosome end (22). At least 100 kb can be copied, resulting in loss of heterozygosity of all markers distal to the point of strand invasion. The RAD51-independent BIR process requires Rad59, the Rad54 homolog Tid1, and the Mre11-Rad50-Xrs2 (MRX) complex (11,32). A striking feature of RAD51-independent BIR initiated at the MAT locus on chromosome III is that the site where repair apparently initiates is far from the DSB site, predominantly making use of a facilitator of BIR (FBI) sequence located more than 30 kb closer to the centromere (23). When RAD51-independent BIR occurs on a plasmid or when a linear DNA fragment lacking a telomere is transformed into budding yeast, there ...

Break-induced replication occurs by conservative DNA synthesis

“…BIR events can best be detected by creating a DSB where just one of the two ends can undergo homology-dependent strand invasion (9,13,18). To model this reaction we designed a chromosomal substrate by inserting a recipient cassette with a 3′ truncated lys2 gene (lys), a 36-bp homothallic switching (HO) endonuclease cut site (HO cs), and KanMX to confer resistance to geneticin (G418), 34 kb from the left telomere of chromosome (Ch) V. There are no essential genes distal to the site of the insertion (13,19).…”

“…However, if coordination of the two ends is not maintained or only one end of the break is available, such as at a critically short telomere, repair can occur by break-induced replication (BIR) (7). In this case, following strand invasion replication occurs to the end of the chromosome to generate a stable repaired product (8,9). This process can cause very long gene conversion tracts and significant LOH, and nonreciprocal translocation if invasion occurs at a dispersed repeated sequence (10).…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…BIR was first described as a RAD51-independent error-prone DSBR pathway and is known to mediate several types of chromosomal instability in yeast [55][56][57][58]. BIR is as processive as conventional chromosomal replication and it therefore probably entails formation of a replication fork at the recombination joint [59] (Fig. 3).…”

“…3). The recent description of a RAD51-dependent form of BIR in S. cerevisiae suggests that BIR may not merely be an error-prone system of DSBR [59,60]. RAD51-dependent BIR might be a true analog of (RecA-dependent) RDR in E. coli.…”

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

“…Third, mice deficient in both NHEJ and p53, develop lymphomas, resulting from rearrangements initiated by V(D)J recombination-activating protein (RAG)-induced DNA cleavage and involving a break-induced replication (BIR) pathway (Vanasse et al, 1999;Difilippantonio et al, 2002;Zhu et al, 2002). BIR is a DSB repair process requiring sequence homology and in part Rad51-dependent in yeast (Kraus et al, 2001;Malkova et al, 2005).…”