Comprehensive analysis of cis- and trans-acting factors affecting ectopic Break-Induced Replication

Abstract

Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating ectopic BIR efficiency, we found that ectopic BIR efficiency is the highest close to chromosome ends. The variations of ectopic BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of ectopic BIR depends on the length of DNA already synthesized. Ectopic BIR is more susceptible to disruption during the synthesis of the first ~35-40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes ectopic BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.

Fig 1. Ectopic BIR efficiency along the right arm of chromosome IV.

A. The BIR assay used in this study is based on the transformation into yeast cells of a linearized chromosome fragmentation vector (CFV). This CFV contains a telomere seed (black arrowhead), the URA3 auxotrophic marker, the centromere from chromosome IV (CEN4) and a region of homology with the genome to initiate BIR (KANMX4 or BUD3). This CFV being deprived of any origin of replication, the only way it can be maintained into the cell is after undergoing BIR to incorporate an origin of replication from the chromosome as well as a telomere to be stabilized. The number of transformants obtained using the linearized plasmid reflects BIR efficiency. Left: the region of homology with the genome is the KANMX4 selection cassette. This cassette has been used to systematically replace individually all the S. cerevisiae ORFs keeping the same orientation of transcription as the endogenous ORFs [31]. Right: the region of homology with the genome encompasses BUD3. B. Ratio between the efficiencies of BIR initiated from the KANMX4 selection cassette from different loci along the right arm of chromosome IV and the efficiency of BIR initiated from BUD3. Mean ratios of five (1,080; 981; 875; 11), four (781; 678; 383; 282; 182) and three (480; 115) independent experiments are shown. Error bars represent standard deviation.

Fig 2

A. Diagram representing the twelve BIR initiating loci used to generate Fig 2B (S3 Table). Top: wild type strains with nine BIR initiating loci on the right arm of chromosome IV. Bottom: translocated strains with three BIR initiating loci IVR-398, IVR-338 and IVR-291 corresponding to the IVR-117, IVR-57 and IVR-10, respectively, from the parental non-translocated strains. B. BIR efficiency modelled as a combination of two exponential functions of the length of DNA to synthesize. BIR efficiency is expressed as the ratio between the number of transformants from the linearized chromosome fragmentation vector and the number of transformants from the circular chromosome fragmentation vector containing an autonomous replicating sequence (ARS). The two red curves represent exponential law fits. See main text for details.

Fig 3. Ectopic BIR efficiency in translocated strains where the distance between the BIR initiating loci and the telomere was modified.

A. The translocation exchanges the last 101 kb of the right arm of chromosome VII with the last 10 kb of the right arm of chromosome IV, increasing by 91 kb the length of DNA to synthesize for BIR initiated at the IVR-117, IVR-57 and IVR-10 loci. WT: reference (wild type) strain. B. The translocation exchanges the last 12 kb of the right arm of chromosome XVI with the last 113 kb of the right arm of chromosome IV. This decreases by 101 kb the length of DNA to synthesize for BIR initiated at the IVR-117 locus, but does not modify the distance to the telomere of the IVR-57 and IVR-10 loci. The WT values are the same in the two graphs and represent the means of 49 independent experiments. Values for the translocated strains represent the means of three independent experiments. Error bars represent standard deviations. Indicated are Student’s t-test p-values <0.05 when comparing a given point from a translocated strain with its equivalent from the parental strain without translocation.

Fig 4. Ectopic BIR efficiency is decreased by cis-acting factors including transcription and heterochromatin of the template chromatid.

A. A chromosome translocation positioned the last 16 kb of the left arm of chromosome VI downstream of the IVR-10 BIR initiating locus. This region contains the THI5, SNZ3 and SNO3 genes whose transcription is active in the absence of thiamine in the growth medium and inactive in its presence. The arrows indicate the transcription orientation of the corresponding genes. Values are the means of three independent experiments in the presence of thiamine and four experiments in the absence of thiamine. Indicated is the Student’s t-test p-value <0.05 when comparing BIR efficiency with or without thiamine in the growth medium. B. BIR efficiency from the IVR-117, IVR-57 and IVR-10 loci in a sir3 null mutant. WT values are identical to those in Fig 3. The sir3 values correspond to the means of five independent experiments. Indicated are Student’s t-test p-values <0.05 when comparing BIR efficiency from a given locus in the mutant strain with the BIR efficiency from the same locus in the WT strain. Error bars represent standard deviations.

Fig 5. Ectopic BIR efficiency is affected by the trans-acting factors Dun1, Rad51, Rad52 and Mph1.

Indicated are Student’s t-test p-values <0.05 when comparing BIR efficiency from a given locus in a mutant strain with the BIR efficiency from the same locus in the WT strain. Error bars represent standard deviations. A. BIR efficiency of the null mutants dun1, sml1 and dun1 sml1 from the IVR-117, IVR-57 and IVR-10 loci. WT values are identical to those in Fig 3. Mutant values are the means of four independent experiments for dun1 and three independent experiments for sml1 and dun1 sml1. B. Similar to A with RAD51 and RAD52 overexpression (OE) from a multicopy vector. Values are the means of three independent experiments for each condition. C. Similar to A with the mph1 null mutant. WT values are identical to those in Fig 3. Values for the mph1 strain are the means of seven independent experiments for IVR-117 and IVR-10 and six independent experiments for IVR-57.

Fig 6. Ectopic BIR efficiency in the absence of Srs2.

A. Parental strain configuration. The IVR-10 locus overlaps a Ty1 LTR (YDRWdelta31). The telomeric region of the right arm of chromosome IV TEL04R is composed of an X element core sequence (XC), a ca. 475 bp sequence common to all chromosome ends; X element combinatorial repeats (XR); an internal stretch of telomeric repeats (TR); a long Y’ element (Y’-L) and a terminal stretch of telomeric repeats (right arrowhead). In the absence of Srs2, BIR efficiency decreases from the IVR-10 locus only. B. The last 113 kb of the right arm of chromosome IV were replaced by reciprocal translocation with the last 12 kb of the right arm of chromosome XVI. The telomeric region of the right arm of chromosome XVI TEL16R is composed of an XC; a short Y’ element (Y’-S) and a terminal stretch of telomeric repeats. The IVR-117 locus is 16 kb away from the telomere in the translocated strain and does not overlap any LTR. In the absence of Srs2, BIR efficiency decreases from the IVR-117 and IVR-10 loci that are located 16 and 10 kb away from the flanking telomeres TEL16R and TEL04R, respectively. C. The last 113 kb of the right arm of chromosome IV were replaced by reciprocal translocation with the last 10 kb of the right arm of chromosome VI. The telomeric region of the right arm of chromosome VI TEL06R is composed of an XC and a terminal stretch of telomeric repeats. The IVR-117 locus is 14 kb away from the telomere in the translocated strain and does not overlap any LTR. In the absence of Srs2, BIR efficiency decreases from the IVR-117 and IVR-10 loci that are located 14 and 10 kb away from the flanking telomeres TEL06R and TEL04R, respectively. D. Effect of ploidy and mating type heterozygosity on BIR efficiency. WT values in panel A are the same as in Fig 3. The srs2 values of panel A and the values of panel C are the means of three independent experiments. The values of panels B and D are the means of four experiments. Indicated are Student’s t-test p-values <0.05 when comparing BIR efficiency from a given locus in a mutant strain with the BIR efficiency from the same locus in the relevant reference strain. Error bars represent standard deviations.

Fig 7. PFGE and Southern blot of BIR products initiated at the IVR-10 locus in the absence of Srs2.

Ethidium bromide stained gels (left) and corresponding radioactive signal from Southern blot using a URA3 probe specific of the chromosome fragmentation vector (right) are shown. Arrowheads indicate chromosome fragments (CF) of abnormal size resulting from BIR initiated by the chromosome fragmentation vector. Gross chromosomal rearrangements (GCR) are indicated by arrows. Roman numbers on the left correspond to S. cerevisiae chromosome numbers.