The SIR2/3/4 complex promotes efficient break-induced replication by stabilizing recombination intermediates
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Homologous recombination (HR) is one of the most effective pathways a cell can employ to repair DNA double strand breaks (DSBs). While most of the HR machinery has been characterized, less is known about how chromatin is reorganized and participates in repair. Here, we use a break-induced replication (BIR) assay, which measures conservative copying of a donor chromosome arm onto a single-ended recipient sequence, to explore a direct role for the SIR2/3/4 (SIR) silent chromatin complex in HR. Specifically, we measure an early step of BIR, D-loop extension, and simultaneously monitor localization of the SIR complex at the initiating DSB and an ectopic donor locus. Interestingly, we observe an ∼four- fold increase in Sir2 and Sir3 protein levels upon DSB induction and detect an enrichment of all three SIR complex subunits at the DSB and donor loci during periods of active D-loop extension. ChIP-seq experiments reveal enrichment of SIR complex subunits that extend up to 15 kb from the DSB site. This enrichment is substantially reduced in rad51Δ and rad52Δ mutants that show defects in BIR. Lastly, we show that sir mutations confer slowed D-loop extension kinetics between divergent substrates and increase the rate of mutation during BIR. These data are consistent with a model in which the SIR complex acts to stabilize the extending D-loop during BIR, leading to more efficient, and thus less mutagenic repair.
Author Summary
Double-strand breaks (DSBs) are among the most dangerous types of DNA lesions that occur in a cell; if unrepaired, they can cause cell death and deleterious chromosomal rearrangements. Homologous recombination (HR) is one of the most effective pathways to repair DSBs. While the machinery involved in HR has been characterized, less is known about how HR acts within a chromatin environment which contains DNA packaged into a compact state. In baker’s yeast transcriptionally inactive “silent” chromatin is present at a limited number of domains in the cell. Bound to and regulating the transcriptional inactivity of these domains is the SIR complex. Curiously, various components of the SIR complex have been shown to localize to HR repaired DSBs. However, little is known about how they participate in HR. In this study we use a break-induced replication (BIR) assay, which measures conservative copying of a donor chromosome onto a single-ended recipient sequence, to characterize roles for the SIR complex in HR. We detect an enrichment of all three SIR complex subunits at the recipient and donor loci during periods of active repair. ChIP-seq, a method that detects protein-DNA interactions genome-wide, reveals enrichment of SIR complex subunits up to 15 kb from each side of the DSB site. This enrichment is significantly reduced in mutants that show defects in BIR. We assess the functional consequences of the loss of the SIR complex, showing that sir mutations slow early steps in BIR and increase the rate of mutation. Our data are consistent with a model in which the SIR complex acts to stabilize early intermediates in BIR, leading to more efficient, and thus less mutagenic repair.
