Pathway utilization in response to a site-specific DNA double-strand break in fission yeast

Abstract

We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe. Tech nology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch(16), using the Saccharomyces cerevisiae HO-endonuclease and its target site, MATa. DSB repair in this context was predominantly through interchromosomal gene conversion. We found that the homologous recombination (HR) genes rhp51(+), rad22A(+), rad32(+) and the nucleotide excision repair gene rad16(+) were required for efficient interchromosomal gene conversion. Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3(+). Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55Delta background involving ku70(+) and rhp51(+). Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70Delta and lig4Delta non-homologous end joining (NHEJ) mutant backgrounds compared with wild type. Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion. These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways.

Break-induced ATR and Ddb1–Cul4Cdt2 ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

“…Quantitative analysis of break repair in rad3D revealed a defect in HR repair, resulting in a significantly reduced level of GC (26%, P = 0.001) and a concomitant increase in Ch 16 loss (42%, P = 0.03) and LOH (27%, P = 0.0003) compared with wild type ( Fig. 6C; Prudden et al 2003), reminiscent of the repair profile observed in ddb1D and cdt2D. Furthermore, analysis of break repair following deletion of spd1 + in a rad3D background partially restored GC levels (37%, P = 0.05) and reduced levels of minichromosome loss (38%, P = 0.5) and LOH (20%, P = 0.01) compared with rad3D (Fig.…”

“…2B; Tinline-Purvis et al 2009). Following break induction, wild-type cells predominantly repair the break by HR repair (61%), resulting in arg + hyg S ade + his + colonies in which the MATa break site and adjacent Hyg R marker are lost through gene conversion (GC) using the endogenous ChIII as a repair template (Prudden et al 2003). In contrast to wild type, significantly reduced levels of GC were observed in both ddb1D (34%, P = 0.005) and cdt2D (33%, P = 0.02) backgrounds following break induction, an outcome indicative of inefficient HR.…”

“…FACS analysis indicated that cdc22-M45 cells exhibited a 2C peak, and were thus predominantly in G2 at the time of DSB induction during the DSB assay (Supplemental Fig. 4; Prudden et al 2003).…”

“…The DSB assay was performed as described previously (Prudden et al 2003). The percentage of colonies undergoing NHEJ/SCC (arg + Hyg R ade + his + ), GC (arg + Hyg S ade + his + ), minichromosome loss (arg À Hyg S ade À his À ), or LOH (arg + Hyg S ade À his À ) was calculated.…”

“…FACS analysis indicated that cdc22-M45 cells exhibited a 2C peak, and were thus predominantly in G2 at the time of DSB induction during the DSB assay (Supplemental Fig. 4; Prudden et al 2003).A role for the Ddb1-Cul4 Cdt2 ubiquitin ligase complex in gap filling of break-induced resected ssDNAReduced nucleotide pools observed in a ddb1D background would be expected to affect DNA synthesis steps Cold Spring Harbor Laboratory Press on May 10, 2018 -Published by genesdev.cshlp.org Downloaded from required for gap filling and efficient GC following a DSB. The extent of ssDNA generated through resection would dictate the length of new DNA synthesis required to fill the gaps following annealing of ssDNA ends.…”