Emerging non-canonical roles for the Rad51–Rad52 interaction in response to double-strand breaks in yeast

Abstract

DNA double-strand break repair allows cells to survive both exogenous and endogenous insults to the genome. In yeast, the recombinases Rad51 and Rad52 are central to multiple forms of homology-dependent repair. Classically, Rad51 and Rad52 are thought to act cooperatively, with formation of the functional Rad51 nucleofilament facilitated by the mediator function of Rad52. Several studies have now identified functions for the interaction between Rad51 and Rad52 that are independent of the mediator function of Rad52 and affect a seemingly diverse array of functions in de novo telomere addition, global chromosome mobility following DNA damage, Rad51 nucleofilament stability, checkpoint adaptation, and microhomology-mediated chromosome rearrangements. Here, we review these functions with an emphasis on our recent discovery that the Rad51-Rad52 interaction influences the probability of de novo telomere addition at sites preferentially targeted by telomerase following a double-strand break (DSB). We present data addressing the prevalence of sites within the yeast genome that are capable of stimulating de novo telomere addition following a DSB and speculate about the potential role such sites may play in genome stability.

When the Ends Justify the Means: Regulation of Telomere Addition at Double-Strand Breaks in Yeast

“…More recently, a total of seven additional hotspots of dnTA have been identified in yeast. Induction of HO cleavage at least 2 kb distal to these sequences results in telomere addition within the hotspot at a frequency ∼200-fold higher than in neighboring sequences, ruling out a model in which telomere addition at the hotspot is a simple consequence of chromosome fragility (Obodo et al, 2016;Ngo et al, 2020). We call such endogenous sequences Sites of Repair-associated Telomere Addition, or SiRTAs.…”

Hotspot ofde novotelomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions

“…The observation that many of the telomere addition events associated with extrachromosomal SUL1 fragments cluster within a small (∼100 bp) region is reminiscent of sequences in the yeast genome that are hotspots of de novo telomere addition (termed SiRTAs or Sites of Repair-associated Telomere Addition) (Stellwagen et al 2003; Obodo et al 2016; Hoerr et al 2021). SiRTAs contain strand-specific, TG-rich sequence motifs (Obodo et al 2016; Ngo et al 2020). These motifs can provide a substrate for the acquisition of telomeric sequences if they become exposed in single-stranded DNA either as a result of 5’ end resection following a DSB or at nicks in single-stranded gaps.…”

“…Like telomeres, SiRTAs have an asymmetric sequence pattern in which one strand is relatively rich in T and G nucleotides (Obodo et al 2016; Ngo et al 2020). For telomerase to act upon a SiRTA, this TG-rich strand must be exposed in single-stranded DNA.…”

“…To determine the ability of this 718 nt sequence to stimulate telomere addition, we took advantage of an assay in which the sequence of interest is integrated on the left arm of chromosome VII, replacing sequences between chromosome VII coordinates 18086 and 18108 (Ngo et al 2020). This strain (Lydeard et al 2010; Obodo et al 2016) contains a single recognition site for the yeast homothallic switching (HO) endonuclease that allows regulated generation of a double-strand break approximately 2 kb distal to the sequence being tested (Figure 3A).…”

“…Creation of the ars228 deletion has been described (Brewer et al 2015). Strains for the HO cleavage assay were generated as described in Ngo et al 2020. In brief, the HO endonuclease recognition sequence was integrated on chromosome VII by one-step gene replacement using plasmid pHO invC (based on JH2017; gift of J. Haber). The URA3 gene was integrated by one-step gene replacement using pRS306 (Sikorski and Hieter 1989) as template.…”