Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated.

Abstract

HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways. In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions. Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product. Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb. Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded. Gap repair processes are independent of distance but are reduced in rad52 mutants and in Gl-arrested cells.In both mitotic and meiotic cells, double-strand breaks (DSBs) can initiate homologous recombination events. In the yeast Saccharomyces cerevisiae, this process has been characterized by analyzing the outcome of repair of DSBs induced by X rays, transformation of linear substrates into cells, and introduction of site-specific lesions in both mitotic and meiotic cells (1,18,36,37,44,54 1A) proposes that a DSB or gap is repaired by a gene conversion event involving the concerted participation of both broken ends, which invade and copy DNA from an intact, homologous donor copy. More recent considerations have prompted a modification of this model to include the creation of extensive single-stranded regions by 5'-to-3' exonucleolytic digestion of the DSB (55,63). This model predicts that gene conversion can occur both with and without an accompanying crossing over and that these two products occur with equal frequencies (Fig. 1A).Similar recombination events have been described for mammalian cells. Linearized DNA molecules can be used to effect site-directed gene replacement (13,27,50) and gap repair of transformed DNA (2). Recently, a system that carries out gap repair in vitro has been developed; however, some of the products obtained had not been predicted by a simple gap repair model (15). A number of studies of recombination in mammalian cells suggest that there is an alternative pathway of recombination, termed single-strand annealing (SSA, Fig. 1B) (3,6,(21)(22)(23)61). In this pathway (Fig. 1B), deletions are formed between sequences in direct orientation. A DSB produced between the directly repeated sequences is attacked by a 5'-to-3' exonuclease whose degradation produces complementary single strands which then anneal. This mechanism also accounts for products resembling gene conversions that have arisen by the interaction of several DNA molecules (22 (29)(30)(31).With yeast, the molecular events associated with recombinational repair of DSBs can be observed in vivo by physically monitoring DNA after the synchronous induction of a DSB. With mitotic cells, it has been possible to carry out a detailed analysis of DSB repair by using two highly efficient site-specific endonucleases: the HO endonuclease, whic...

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…We hypothesized that break resection would normally be chased and terminated by DNA re‐synthesis which would have a much faster rate than the processing [140 kb/h for conventional replication (Raghuraman et al , 2001), but could be slower during repair synthesis, vs. 4 kb/h for resection (Fishman‐Lobell et al , 1992)]. In fact, by comparing the reconstitution of fragments S2 and L (Fig 6B), we could estimate the rate of dsDNA restoration in srs2 Δ.…”

“…During HR and BIR, Rad52/51/55/57 promote homology search and invasion of intact donor dsDNA by the processed broken end to initiate repair (Anand et al , 2013; Symington et al , 2014). In contrast, SSA does not require DNA external to the broken chromosome as homologous sequences on either side of the break provide complementarity between the processed ends and Rad52, but not Rad51/55/57, catalyse the strand annealing (Fishman‐Lobell et al , 1992; Ivanov et al , 1996). …”

Does age influence loss of heterozygosity?

“…Previous work in young cells has demonstrated that different repair pathways can compete for the same DNA lesion (Fishman-Lobell et al, 1992). In effect the DNA end produced at a DSB site is a substrate for which various DNA repair proteins compete.…”

“…For example, sister chromatid exchange cannot take place in G1 (there is no sister chromatid). In fact, homologous recombination appears to be repressed in G1 (Fishman-Lobell et al, 1992) and NHEJ is preferentially used in this stage of the cell cycle (Aylon and Kupiec, 2005). Cell-type specific regulation of the DNA damage repair method has also been demonstrated.…”

“…For example haploid yeast exert mating type control of NHEJ (Frank-Vaillant and Marcand, 2001). In addition alternative pathways for DSB repair can effectively compete with one another at the same break site (Fishman-Lobell et al, 1992;Tutt et al, 2001). As shown in budding yeast and cultured mammalian cell lines, if one repair pathway within a cell is compromised, whether due to the conditions of the DSB or dysfunction in components of the pathway, other repair pathways are able to compete for the DNA substrate and change the predominate form of repair used.…”