Double-strand breaks arising by replication through a nick are repaired by cohesin-dependent sister-chromatid exchange

Abstract

Molecular studies on double-strand break (DSB) repair in mitosis are usually performed with enzymatically induced DSBs, but spontaneous DSBs might arise because of replication failures, for example when replication encounters nicks. To study repair of replication-born DSBs, we defined a system in Saccharomyces cerevisiae for the induction of a site-specific single-strand break. We show that a 21-base pair (bp) HO site is cleaved at only one strand by the HO endonuclease, with the resulting nick being converted into a DSB by replication during the S phase. Repair of such replication-born DSBs occurs by sister-chromatid exchange (SCE). We provide molecular evidence that cohesins are required for repair of replication-born DSBs by SCE, as determined in smc3, scc1 and scc2 mutants, but not for other recombinational repair events. This work opens new perspectives to understand the importance of single-strand breaks as a source of recombination and the relevance of cohesion in the repair of replication-born DSBs.

Figure 1

Breaks at the 21-bp HO site are primarily single-strand nicks. (A) Scheme of plasmid pRS316-TINV before and after the HO-induced DSB. Lines with asterisks indicate the LEU2 probe used for hybridization experiments. (B) Kinetics of HO cleavage as assayed by native and alkaline gel electrophoresis of DNA from asynchronous (ASYNC) and G1-arrested cultures of WS-bar1 strains. DNA samples were taken at the indicated time after HO induction and cut with XhoI and SpeI before electrophoresis. Fluorescence-activated cell sorting pattern of cultures and quantification data of nicks and DSBs are shown. The percentage of plasmids containing nicks was calculated as twice the difference between the number of broken molecules observed in the alkaline and native gels and normalized with respect to the total DNA fragments. Plasmids containing DSBs were determined directly from the native gels. DSBs, double-strand breaks; Gal, galactose.

Figure 2

Nicks are made in both strands. Breaks in each DNA strand were determined in alkaline gels by hybridization of DNA from G1-arrested cells with LEU2 single-stranded specific probes. For other details, see Fig 1. Gal, galactose.

Figure 3

HO-induced double-strand breaks at the 21-bp HO site are formed after DNA replication. (A) Fluorescence-activated cell sorting analysis of the WS-bar1 cells arrested in G1 with α-factor and at different periods after release. (B) Kinetics of DSB formation (native gel) after release from G1 arrest, as determined by Southern blot analysis. After 2 h of HO induction in galactose (Gal), WS-bar1 G1-arrested cells were released from α-factor in the presence or absence of 50 mM HU. Quantification data are plotted at the bottom. For other details, see Fig 1. DSB, double-strand break; HU, hydroxyurea.

Figure 4

Cohesion is required for sister-chromatid exchange but not for intrachromatid recombination. (A) Schemes of plasmid pRS316-TINV and the intermediates produced by unequal SCE and ICR. Each band detected after XhoI–SpeI cleavage, using the LEU2 probe shown in Fig 1, is indicated with its corresponding size. Note that ICR events observed in these assays occur by break-induced replication, and gene conversions are not detected. (B) Kinetics of DSB formation, SCE and ICR intermediates in isogenic wild-type (wt) and smc3-42 strains. (C) Kinetics of DSB formation, SCE and ICR intermediates in isogenic scc1-73 and scc2-4 strains. Wt and smc3-73 data, taken from (B), are plotted for comparison. Cultures were incubated at restrictive temperature (37°C) for 30 min before HO induction. SCE levels were calculated as the ratio between the 4.7-kb band and the total plasmid DNA. ICR levels were calculated by subtracting the density value of the 4.7-kb band from that of the 2.9-kb band. For other details, see Fig 1. DSB, double-strand break; ICR, intrachromatid recombination; SCE, sister-chromatid exchange.

Figure 5

Cohesins are required for equal sister-chromatid exchange. (A) Schematic representation of the monomeric plasmid pCM189-leu2HOr, containing no DNA repeats, and the dimer obtained by equal SCE. (B) Kinetics of DSB formation and equal SCE intermediates in isogenic wild-type (wt) and smc3-42 strains. Plasmid DNA was not digested before electrophoresis. lM, linear monomers; rD, relaxed dimers; rM, relaxed monomers; scD, supercoiled dimers; scM, supercoiled monomers. Quantification of dimers (rD and scD) related to total plasmid DNA is shown. For other details, see Fig 1. DSB, double-strand break; SCE, sister-chromatid exchange.

Figure 6

Model to explain the role of cohesins in the repair of double-strand breaks formed after replication forks encounter single-strand DNA breaks. Double-ended DSBs could be formed after replication through a nick in two ways depending on whether the nick is encountered at the lagging (left) or the leading strand (right). Cohesins (thin grey ovals), which are additionally loaded in the proximity of the DSB, favour HR with the sister. DSB, double-strand break; HR, homologous recombination.