Timing is everything: cell cycle control of Rad52

Abstract

Regulation of the repair of DNA double-strand breaks by homologous recombination is extremely important for both cell viability and the maintenance of genomic integrity. Modulation of double-strand break repair in the yeast Saccharomyces cerevisiae involves controlling the recruitment of one of the central recombination proteins, Rad52, to sites of DNA lesions. The Rad52 protein, which plays a role in strand exchange and the annealing of single strand DNA, is positively regulated upon entry into S phase, repressed during the intra-S phase checkpoint, and undergoes posttranslational modification events such as phosphorylation and sumoylation. These processes all contribute to the timing of Rad52 recruitment, its stability and function. Here, we summarize the regulatory events affecting the Rad52 protein and discuss how this regulation impacts DNA repair and cell survival.

Figure 1

Repair of a DSB proceeds according to cell cycle stage. In G1, cells have a single copy of each chromosome (light blue and light green). If a break occurs in G1, the cell repairs the DSB by NHEJ, directly resealing the DNA ends (top). In G2, after chromosomes have been replicated, there is a sister chromatid, an identical copy of each original chromosome (dark blue and dark green, bottom). When a DSB occurs in G2, it is normally repaired by HR using either the sister chromatid or a homologous chromosome as a template. Repair from the sister (I) results in restoration of the exact information lost at the break site. Repair from the homolog (II), however, may lead to loss of heterozygosity if accompanied by a crossover.

Figure 2

Models of homologous recombination. DSBs can be repaired using the homologous recombination machinery in a variety of ways. The DNA ends are first processed into 3' ssDNA tails. These tails invade a homologous template (red) priming new DNA synthesis (dashed line). Three possible outcomes from this invasion are shown. A) In canonical DSBR, both the initial invading strand and the captured second end anneal to the homologous template and prime new DNA synthesis, resulting in a double Holliday junction that can be resolved by nucleases into crossover or non-crossover products (non-crossover product shown). B) Alternatively, after the single ssDNA tail invades the homologous template, a round of DNA synthesis is primed from the 3' end (dashed red line). Synthesis-dependent strand annealing (SDSA) occurs when the invading strand, along with the newly synthesized segment, is unwound by a helicase and annealed with the other resected end. C) In break-induced replication (BIR), one end of the DSB is lost and the remaining end invades the homologous template priming DNA synthesis to the end of the chromosome.

Figure 3

Regulation of HR. Recruitment of the HR machinery to a DSB is regulated by both the major cell cycle kinase CDK1 and the checkpoint kinase Mec1. CDK1 phosphorylation is marked as a yellow circle and Mec1 phosphorylation is marked as a yellow star. A) Once a DSB is detected, the DNA ends are resected forming 3' ssDNA tails by multiple nucleases that are positively regulated by CDK1/B type cyclin kinase activity. RPA binds the ssDNA and recruits the ATR-ATRIP homolog Mec1-Ddc2 and the 9-1-1 complex comprised of Ddc1, Mec3 and Rad17 (indicated by Ddc1). Finally, Rad52 catalyzes the formation of a Rad51 nucleoprotein filament along the ssDNA before HR can proceed. B) The intra-S phase checkpoint proteins Mrc1, Tof1 and Csm3 travel with the fork during normal replication. In response to DNA damage, the replication fork stalls, activating Mec1 which in turn phosphorylates Mrc1. Phosphorylated Mrc1 promotes stable fork pausing and contributes to Mec1 retention at the fork. In the absence of Mec1, the replisome is not stable when the fork pauses or stalls, leading to the uncoupling of the MCM helicase and the polymerase (grey) and fork collapse (bottom right). The DNA replication clamp PCNA is the circle adjacent to MCM2-7. Rad52 is recruited to the collapsed fork and HR restarts replication by one-end invasion of the intact DNA molecule (here shown as lagging strand invasion of the leading strand template). C) In repetitive sequences (indicated by green arrows) the Smc5/6 complex is recruited to DSBs along with the DSBR machinery, shown in A, to mediate repair. Smc5/6 and the sumoylation state of Rad52 affect whether repair deletes or retains DNA sequences between repeats (purple triangles) during direct repeat recombination.