RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion

Abstract

Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time. However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms. Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome. Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G(2)/M DNA damage checkpoint. RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process. RAD51-dependent BIR occurs efficiently in G(2)-arrested cells. Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.

FIG. 1.

Experimental system to study BIR and GC. (A) Chromosome III in diploid strain MLN134, used to study kinetics of GC. Positions of restriction endonuclease recognition sites are indicated as follows: S, StuI; Bg, BglII; K, KpnI; B, BamHI. p1, p2, and p3 indicate positions of primers that were used for PCR analyses of intermediates of GC and BIR. (B) Chromosome III in diploid strain MY006, used to study BIR. The MATa-containing copy of chromosome III is truncated by insertion of a LEU2 gene fused to telomere sequences. All abbreviations are similar to those in panel A. (C) Illustration of GC repair of MY006. (D) BIR repair of MY006. (E) Chromosome loss when the HO cut in MY006 is not repaired. In all cases, repair occurring in just one chromatid is illustrated for simplicity. In a case of DSB repair in G₂, the colony phenotype depends on independent repair of two chromatids.

FIG. 2.

BIR and GC analyzed by pulse-field gel electrophoresis. DNA was prepared for pulse-field gel (CHEF) electrophoresis at intervals after induction of a DSB at MAT. Southern blots were probed with ADE1, which hybridizes to chromosome I (Ch I) and to hml::ADE1 on chromosome III. (A) GC in strain MLN134. The initial appearance of product is indicated with an arrowhead. (B and C) A diploid carrying a truncated chromosome III (Ch IIITr) yields both GC and BIR in strain MY006 (B) and strain AM792 (C). The first significant increase in product formation is indicated with an arrowhead. (D) Kinetics of accumulation of GC in strain MLN134 (□) and BIR in strain MY006 (Δ) and strain AM792 (•).

FIG. 3.

Southern blot analysis of GC and BIR. (A) DNA digested with BglII to analyze GC in strain MLN134. The initial product appearance is shown by an arrowhead. (B and C) DNA digested with KpnI was analyzed for GC and BIR in strain MY006 (B) and strain AM792 (C). Blots were probed with a 320-bp fragment proximal to MAT locus. (D) The same blot as shown in panel C but probed with a LEU2-specific probe. (E) The appearance of GC and BIR products for AM792 is compared with GC results for MLN134. The rate of appearance of GC in AM792 is apparently retarded compared to that of MLN134 because the maximum level of GC is overestimated, as cells that repair DSBs by GC resume division whereas repair by cells using BIR is delayed. (F) Appearance of GC and BIR in MY006 compared with that of GC in MLN134.

FIG. 4.

Timing of strand invasion during BIR versus GC. (A) PCR detection of a strand invasion and extension intermediate in DSB repair. The example shown is for strain AM792, where the left end of the DSB is perfectly homologous to the donor template. In strain MY006, the first 650 bp of the left end of the DSB are not homologous to the MATα-inc donor sequence. (B) PCR analysis of strand invasion and later steps of repair in diploid strain MY006 by use of primers p1 and p2 (Fig. 1B). The arrow indicates a position of the 5-kb PCR product corresponding to the strand invasion product formed by BIR. (C) PCR analyses of samples of strain AM792 by use of primers p1 and p2 (Fig. 1B) reveals a 5-kb product corresponding to the strand invasion product formed by BIR. (D) Timing of strand invasion during GC as determined by PCR analysis of samples from MLN134 (Fig. 1A). Use of primers p1 and p3 (Fig. 1A) revealed a 4-kb product corresponding to the strand invasion product formed by GC. (E) Accumulation of BIR strand invasion products in MY006 and AM792. The PCR data presented in panels A and B are quantitative for the initial time points up to 5 h. For the quantitation of the amounts of products at the later time points we used PCR performed on diluted DNAsamples. The results presented in panel C are quantitative only for the 2-h time point, as the amount of product was saturated for all later time points.

FIG. 5.

BIR in strain MY006 cells arrested in G₂ by nocodazole. A Southern blot of the appearance of BIR products on a CHEF gel probed with ADE1, which hybridizes to chromosome I and to the HO-cut truncated chromosome III, is shown. A BIR product that is the same size as an intact chromosome III and hybridizes also to a THR4 probe (not shown) is indicated as Ch III BIR. A later-appearing translocation that does not hybridize with THR4 is indicated as Tx.

FIG. 6.

Rate of replication associated with BIR. Accumulation of products corresponding to the beginning and to the end of BIR in strain MY006 is shown. Panels A, B, and C show the results of three independent experiments. In experiments shown in panels A and B, samples were taken every 30 min from initially exponential cultures of strain MY006 undergoing BIR. DNA samples were analyzed by CHEF similarly to the analysis described for Fig. 2B (finish) or were digested by BamHI-StuI and analyzed by Southern blotting (start). In the experiment represented by panel C, samples were taken at 1-h intervals and Southern analyses were performed on DNA samples digested by KpnI.

FIG. 7.

FACS analysis of strains undergoing DSB repair after HO cleavage. Repair in strain MLN134 occurs predominantly by GC, whereas repair in strain MY006 occurs predominantly by BIR. Delayed cell cycle progression in strain AM792, in which sequences on both sides of the cleavage site are fully homologous, is shown.