From the Cover: mitotic gene conversion events induced in G1-synchronized yeast cells by gamma rays are similar to spontaneous conversion events

Abstract

In a previous study, we mapped spontaneous mitotic reciprocal crossovers (RCOs) in a 120-kb interval of chromosome V of Saccharomyces cerevisiae. About three-quarters of the crossovers were associated with gene conversion tracts. About 40% of these conversion tracts had the pattern expected as a consequence of repair of a double-stranded DNA break (DSB) of an unreplicated chromosome. We test this hypothesis by examining the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic conversion events associated with spontaneous RCOs, confirming our previous conclusion that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome.

Fig. 1.

Diploid strain used to select and map RCOs. The diploid PG311 has the can1-100 allele on one copy of chromosome V and a replacement of can1 sequences with the ochre-suppressor tRNA gene SUP4-o on the other (5). This strain is also homozygous for the ochre-suppressible ade2-1 mutation. The starting diploid is Can^S and Ade⁺; strains with an unsuppressed ade2-1 mutation form red colonies (3). An RCO between the centromere and can1-100/SUP4-o can result in two Can^R cells (rectangles at Bottom). Subsequent growth of these cells results in a red/white sectored colony. There are many single-nucleotide polymorphisms distinguishing each homologue (indicated by red and black circles) that can be used to map the position of the RCO.

Fig. 2.

RCOs and gene conversion events associated with G2- and G1-induced DSBs. The homologues containing the SUP4-o and can1-100 alleles are shown in black and red, respectively, with circles indicating polymorphic sites. Dotted boxes mark gene conversion tracts and X's show the position of the RCO. (A) The 3:1 conversion tract. Recombination is initiated by a DSB on one of the black chromatids. The associated 3:1 gene conversion involves the broken chromatid receiving information from the red chromatid as observed in many studies of induced DSBs (1). Two linked markers are transferred as shown by the horizontal arrows. (B) The 4:0 conversion tract. Recombination is initiated by a DSB in an unreplicated black chromosome that is then replicated to yield two broken chromatids. The repair of one chromatid is associated with an RCO and conversion of two polymorphisms. The repair of the second DSB is not associated with an RCO but the same two sites are converted, yielding a 4:0 conversion event. (C) The 3:1/4:0 hybrid tract. The pattern of DSB formation and conversion is similar to that in Fig. 2B, except that the conversion tract associated with the second repair event is short, generating a hybrid 3:1/4:0 conversion.

Fig. 3.

Mapping of RCOs and associated conversions in G1- and G2-irradiated cells. The markers are shown at the top with X's and O's indicating the absence and presence of diagnostic restriction sites, respectively. The thick vertical dotted lines mark the divisions that were used in the analysis of the distribution of recombination events (described in the text). Numbers at the top of the figure are the Saccharomyces Genome Database (SGD) coordinates of the markers. Conversion events are shown as horizontal lines with 3:1 events, 4:0 events, and 3:1/4:0 hybrid events indicated by thin lines, thick lines, and hybrid thin/thick lines, respectively. The color of the line shows which chromosome was the donor in the conversion event. Braces indicate complex conversion events and green X's represent RCOs that are not associated with an observable conversion tract. (A) Mapping of 29 RCO events in G1-irradiated cells. (B) Mapping of 31 RCO events in G2-irradiated cells.

Fig. 4.

Mechanism for generating a class 1 complex conversion event by a double repair event. As shown in Fig. 2A, if the event initiates on the SUP4-o-containing chromosome, the sector that is homozygous for SUP4-o will be heterozygous for the marker and the sector that is homozygous for can1-100 will be homozygous. In class 1 complex events, this expectation is violated, because the sector that is homozygous for SUP4-o is homozygous for the marker in the 3:1 portion of the hybrid tract. This pattern is readily explained by a double repair event of a G1-associated DSB. Repair of the first DSB is associated with the RCO and conversion of two markers. Repair of the second DSB is unassociated with a crossover but involves conversion of three markers, two on one side of the DSB and one on the other.