Interaction of yeast Rad51 and Rad52 relieves Rad52-mediated inhibition of de novo telomere addition

Abstract

DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repairassociated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Association of the single-stranded telomere binding protein Cdc13 with a SiRTA is required to stimulate de novo telomere addition. Because extensive resection must occur prior to Cdc13 binding, we utilized these sites to monitor the effect of proteins involved in homologous recombination. We find that telomere addition is significantly reduced in the absence of the Rad51 recombinase, while loss of Rad52, required for Rad51 nucleoprotein filament formation, has no effect. Deletion of RAD52 suppresses the defect of the rad51Δ strain, suggesting that Rad52 inhibits de novo telomere addition in the absence of Rad51. The ability of Rad51 to counteract this effect of Rad52 does not require DNA binding by Rad51, but does require interaction between the two proteins, while the inhibitory effect of Rad52 depends on its interaction with Replication Protein A (RPA). Intriguingly, the genetic interactions we report between RAD51 and RAD52 are similar to those previously observed in the context of checkpoint adaptation. Forced recruitment of Cdc13 fully restores telomere addition in the absence of Rad51, suggesting that Rad52, through its interaction with RPA-coated single-stranded DNA, inhibits the ability of Cdc13 to bind and stimulate telomere addition. Loss of the Rad51-Rad52 interaction also stimulates a subset of Rad52-dependent microhomology-mediated repair (MHMR) events, consistent with the known ability of Rad51 to prevent single-strand annealing.

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

“…Cdc13 associates with a resecting chromosome break even in regions where “ideal” Cdc13 binding sites are not present ( Oza et al, 2009 ), suggesting that Cdc13 binds with low affinity at multiple sites or that other interactions facilitate association with ssDNA. For example, proteins such as RPA and Rad51 influence the recruitment of Cdc13 with DNA ends and the outcome of DNA repair ( Epum et al, 2020 ).…”

“…Mutations that eliminate Cdc13 binding reduce the frequency of dnTA, while mutations that improve Cdc13 affinity increase telomere addition. Replacement of the Stim sequence with the Gal4 upstream activating sequence reduces dnTA, but SiRTA activity is restored by expression of a fusion between Cdc13 and the Gal4 DNA-binding domain ( Obodo et al, 2016 ; Epum et al, 2020 ). In contrast, similar artificial recruitment of the double-stranded telomeric DNA-binding protein Rap1 has no effect.…”

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage

“…However also telomerase can act at DSB, which should be avoided to preserve genome stability. Rad52 is a critical protein for HR in yeast [ 23 , 70 ]. We examined whether the enrichment of Est2 is altered in the absence of Rad52.…”

“…In addition to Cdc13 and Ku70, other proteins and mechanisms have been postulated to regulate the recruitment of Est2 to telomeres. Among them are Pif1 [28,29,31], Mlh1 [49,50], R-loop formation, and Telomeric repeat-containing RNA (TERRA) [51][52][53], RNase P components [13], and Rad51-Rad52 [23]. To reveal if one of these potentially regulatory factors contributes to Est2-binding, we monitored Est2-binding by ChIP-seq in the absence of these factors.…”

“…De novo telomere addition by telomerase at DNA double-strand breaks (DSB) is hazardous for the cell, because all genetic information distal to the DSB is lost [16][17][18][19]. Studies in yeast and human suggest that telomerase components are not associated with the telomere throughout the cell cycle and the catalytic subunit of telomerase itself or associated proteins perform a function at internal regions [8,[20][21][22][23][24][25][26][27]. For example, singlemolecule image tracking of human telomerase revealed a three-dimensional diffusion model wherein telomerase makes multiple transient and stable contacts with telomeres during different cell cycle phases [8,27].…”