Properties of Mitotic and Meiotic Recombination in the Tandemly-Repeated CUP1 Gene Cluster in the Yeast Saccharomyces cerevisiae

Abstract

In the yeast Saccharomyces cerevisiae, the genes encoding the metallothionein protein Cup1 are located in a tandem array on chromosome VIII. Using a diploid strain that is heterozygous for an insertion of a selectable marker (URA3) within this tandem array, and heterozygous for markers flanking the array, we measured interhomolog recombination and intra/sister chromatid exchange in the CUP1 locus. The rate of intra/sister chromatid recombination exceeded the rate of interhomolog recombination by >10-fold. Loss of the Rad51 and Rad52 proteins, required for most interhomolog recombination, led to a relatively small reduction of recombination in the CUP1 array. Although interhomolog mitotic recombination in the CUP1 locus is elevated relative to the average genomic region, we found that interhomolog meiotic recombination in the array is reduced compared to most regions. Lastly, we showed that high levels of copper (previously shown to elevate CUP1 transcription) lead to a substantial elevation in rate of both interhomolog and intra/sister chromatid recombination in the CUP1 array; recombination events that delete the URA3 insertion from the CUP1 array occur at a rate of >10^-3/division in unselected cells. This rate is almost three orders of magnitude higher than observed for mitotic recombination events involving single-copy genes. In summary, our study shows that some of the basic properties of recombination differ considerably between single-copy and tandemly-repeated genes.

Keywords: meiotic recombination; mitotic recombination; repeated genes; sister chromatid crossovers.

Figure 1

Diploid strains (YZ103 and YZ104) used for the detection of interhomolog recombination between CEN8 and the CUP1 array, and within the CUP1 array. (A) Arrangement of markers on chromosome VIII in YZ103 and YZ104. The centromeres are shown as circles. In both diploids, one homolog has the marker hphMX4 inserted in single-copy sequences at the centromere-proximal end of the CUP1 array, an insertion of URA3 in the middle of the CUP1 cluster, and a cassette with CAN1/kanMX4 markers at the centromere-distal end of the array. Both homologs in YZ103 are derived from the haploid W303-1A and have 2.0-kb CUP1 repeats (Zhao et al. 2014); although there are 18 repeats in the CUP1 cluster of W303-1A, only 10 are shown, each in brackets. In YZ104, one homolog (shown in black) is derived from W303-1A, and the other (shown in blue) is derived from YJM789. Only 16 of the ∼22 1.2-kb CUP1 repeats of this homolog are shown. Both strains are phenotypically Hyg^R 5-FOA^S Can^S Gen^R. (B) Detection of a crossover between CEN8 and hphMX4. After a crossover in this genetic interval (shown as an X), followed by cosegregation of chromatids 1 and 3 into one daughter cell and 2 and 4 in the other, one daughter cell (boxed in thin lines) would have the same phenotype as the parental strain, whereas the other daughter (boxed in thick lines) would be phenotypically distinct (Hyg^S 5-FOA^R Can^R Gen^S). (C) Detection of a crossover between hphMX4 and URA3. A crossover in this interval would produce one daughter cell with the same phenotype as the starting diploid and a second daughter with the unique phenotype Hyg^R 5-FOA^R Can^R Gen^S. (D) Detection of a crossover between URA3 and CAN1/kanMX4. One daughter cell would have the same phenotype as the starting strain, and the second daughter would have the phenotype Hyg^R 5-FOA^S Can^R Gen^S. 5-FOA, 5-fluoroorotic acid; Can, L-canavanine; CO, crossover; Gen, geneticin; Hyg, hygromycin B; ^R, resistant; ^S, sensitive.

Figure 2

Mitotic recombination events leading to loss of URA3 marker and retention of the flanking hphMX4 and CAN1/kanMX4 markers. The chromosomes are shown following DNA replication, and the two homologs are shown in different colors; in this depiction, one homolog has 2-kb CUP1 repeats and the other 1.2-kb repeats. CUP1 repeats are indicated by brackets. The chromosomes of 5-FOA^R daughter cells are outlined by thick lines. (A) Intrachromatid “pop-out” recombination. A crossover occurs within a chromatid, producing a shorter CUP1 array and a plasmid with the URA3 gene and three CUP1 repeats. The cell with the chromosomes outlined with thick lines would be 5-FOA^R. Since each CUP1 repeat has an ARS element, the URA3-containing plasmid would be capable of autonomous replication. It is shown segregated into the daughter cell that also contains an integrated URA3 gene. (B) Unequal sister chromatid crossover. As a consequence of this event, the 5-FOA^R daughter cell would contain a shorter CUP1 array, and the Ura⁺ daughter cell would contain a longer array with two URA3 insertions. (C) Intersister chromatid gene conversion. A DSB (shown as a red arrow) occurs near the URA3 insertion in one chromatid, and is repaired using the sister chromatid as a template. The net result of this event would be a loss of URA3 and one or more CUP1 repeats in one daughter cell with no alteration in the second daughter. (D) Interhomolog gene conversion. As in (C), this event is initiated with a DSB near or within the URA3 insertion. The repair template, however, is a chromatid of the other homolog instead of the sister chromatid. Associated with the loss of URA3 and the loss of some of the 2.0-kb CUP1 repeats, one would expect insertion of one or more 1.2-kb CUP1 repeats derived from the other homolog. 5-FOA, 5-fluoroorotic acid; CO, crossover; DSB, double-stranded DNA breaks; ^R, resistant; Ura⁺, uracil⁺.

Figure 3

Loss of the URA3 insertion by SSA or DNA polymerase slippage. In this diagram, we show the chromatid as double-stranded DNA molecules with the 3′-ends marked by arrows. CUP1 repeats are shown as thick arrows and the URA3 insertion is indicated by a rectangle. (A) SSA resulting in a single-stranded DNA loop. Following a DSB (shown with a thick red arrow), the broken ends are processed by 5′–3′ degradation. The single-stranded CUP1 repeat at the terminus at the right broken end anneals with repeats on the top strand of the left broken end. The resulting intermediate has a single-stranded loop containing the URA3 gene. This loop could be removed by cellular exonucleases (shown with thin red arrows) or the resulting DNA molecule could be replicated without removing the loop. The latter event would result in one daughter molecule that retains the URA3 insertion but has fewer CUP1 repeats, and a second molecule that loses the insertion and several CUP1 repeats. (B) SSA resulting in a single-stranded flap. This mechanism is very similar to that of (A) except for the amount of processing and the pattern of annealing. The resulting intermediate has a single-stranded flap that contains both the URA3 gene and one repeat. Removal of the flap would result in a shorter array that lacks the URA3 insertion. (C) DNA polymerase slippage. During DNA replication, the primer strand (the top strand) dissociates from the template and reassociates beyond the position of the URA3 insertion. The resulting intermediate has a single-stranded loop with the URA3 marker and several CUP1 repeats. Removal of the loop or replication of the intermediate would result in a daughter molecule that lacks the URA3 insertion and several CUP1 repeats. DSB, double-stranded DNA breaks; SSA, single-strand annealing.

Figure 4

Detection of interhomolog gene conversion by PCR. CUP1 repeats are outlined by thick black lines. Sequences in common between the 2.0- and 1.2-kb CUP1 repeats are in blue, sequences unique to the 2.0-kb repeat are in white, and those unique to the 1.2-kb repeat are in red. Primers F1 and R1′ amplify both the 2.0- and 1.2-kb repeats whereas the 4038F and 2172R primers are specific for the 2.0- and 1.2-kb repeats, respectively. (A) 2.0-kb repeat array of W303-1A. Only six repeats of the 18-repeat array are shown. (B) 1.2-kb repeat array of YJM789. Only 11 repeats of the 22-repeat array are shown. (C) An array containing both 2.0- and 1.2-kb repeats. As an example of an interhomolog conversion, we show a 2.0-kb array with an insertion of two 1.2-kb repeats. A PCR reaction with primers 4038F and 2172R would produce a unique 1.2-kb band.

Figure 5

Numbers of remaining CUP1 repeats associated with loss of URA3 on the W303-1A-derived chromosome VIII homolog of YZ104; the starting strain had 18 repeats on this homolog. We isolated 33 independent spontaneous 5-FOA^R Hyg^R Gen^R Can^S derivatives. CUP1 copy numbers were determined by measuring the size of CUP1 cluster by Southern analysis of EcoRI-digested genomic DNA. A hybridization probe specific for the W303-1A type of CUP1 repeat was used. 5-fluoroorotic acid; Can, L-canavanine; Gen, geneticin; Hyg, hygromycin B; ^R, resistant; ^S, sensitive.