Abstract
Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.
Highlights
DNA double-strand break (DSB) is the most dangerous type of DNA damage because its inaccurate repair can lead to chromosomal rearrangements, a hallmark of tumorigenesis and tumor progression
To gain deeper insight into the molecular mechanism of synthesis-dependent strand annealing (SDSA) in mammalian cells, we investigated the role of two potential human orthologs of Srs2, namely, FBH1 and RECQ5, in the formation of NCO products during repair of endonuclease-induced DSBs
The DR-GFP reporter consists of a direct repeat of two mutated GFP alleles: a full length GFP interrupted by a recognition site for the I-SceI endonuclease and an internal GFP fragment that serves as a donor for HRmediated repair of a DSB created by I-SceI in the proximal GFP allele (Figure 1A)
Summary
DNA double-strand break (DSB) is the most dangerous type of DNA damage because its inaccurate repair can lead to chromosomal rearrangements, a hallmark of tumorigenesis and tumor progression. HR is mainly restricted to S phase, peaking in mid-S, and requires an intact homologous sequence to be used as a repair template [1,2,3] It is initiated by nuclease-mediated resection of the DNA ends to generate 30-single-stranded (ss) DNA tails that are coated by the ssDNA-binding protein RPA [4]. In DSBR pathway, the second DNA end is captured by the D-loop to form an intermediate with two Holliday junctions, referred to as double Holliday junction (dHJ) This joint DNA molecule can be either resolved by specialized endonucleases into crossover (CO) or noncrossover (NCO) products or dissolved by the BLMTOPOIIIa-RMI1/2 (BTR) complex, which gives rise exclusively to NCO products [5,6,7]. SDSA yields exclusively NCO products [8]
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