A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation

Abstract

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11(+) gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and gamma-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.

Figure 1

Schematic illustration of construction of the rad11-D223Y mutant by allele replacement method. The plasmid shown at the top (pT7-rad11-D223Y-ura4, see Materials and Methods) contains the ura4⁺ cassette and the rad11-D223Y allele. The position corresponding to the D223Y mutation is denoted by a solid circle. The plasmid linearized by digestion with AatII was transformed into haploid strain JY746 (h⁺ leu1-32 ura4-D18 ade6-M210). The rad11-D223Y mutant was obtained by spontaneous direct-repeat recombination between the rad11⁺ allele and the rad11-D223Y allele.

Figure 2

Spontaneous recombination between the ade6B::ura4⁺::ade6X direct repeats. (A) Schematic illustration of intrachromosomal recombination substrate and recombination products. The strain contains ade6B (solid circle) and ade6X (open circle). The ade6 repeats are separated by a 1.8 kb HindIII region containing ura4⁺ marker. ade6⁺ and ura4⁺ alleles are denoted by a filled-in box. ade⁺ ura⁺ recombinants are referred to as conversion-type, and ade⁺ ura^– as deletion-type recombinants. (B) Effect of rad11-D223Y mutation on the spontaneous rate of adenine prototroph formation. The rates of ade⁺ formation and ade⁺ ura⁺ formation were experimentally determined using isogenic strains of the wild type (TNF79) and rad11 (YO006). The rate (×10^–5) of the formation of ade⁺ ura⁺ recombinants (conversion) and the rate (×10^–5) of the formation of ade⁺ ura^– recombinants (deletion) are shown. The rate of ade⁺ ura^– formation was calculated by subtracting the ade⁺ ura⁺ rate from the ade⁺ (ura^±) rate. The recombination rate per cell division was determined using the median value of the recombination frequency of nine cultures. The bar shows the mean value of three sets of experiments. Standard deviations are shown by error bars.

Figure 3

rad11-D223Y cells are MMS and HU sensitive. (A) The sensitivities of wild-type cells (JY746) and rad11-D223Y cells (YO001) to MMS and HU determined in a spot test. (B) Viability of the wild-type cells and the rad11-D223Y cells in YPAD medium in the presence of 20 mM HU. Standard deviations are shown by error bars. (C) Both the wild-type cells and the rad11-D223Y cells were elongated after 8 h of incubation in YPAD medium in the presence of 10 mM HU. For genotypes, see Table 1.

Figure 4

Epistasis analysis between rad11-D223Y cells and rad50-d cells or pku70-d cells for γ-ray sensitivity. (A) The sensitivities to γ-rays of wild-type cells, JY746 (diamonds), rad11-D223Y cells, YO001 (squares), rad50-d cells, KT120 (triangles) and rad11-D223Y rad50 double mutants, YO003 (circles). (B) The sensitivities to γ-rays of wild-type cells, JY746 (diamonds), rad11-D223Y cells, YO001 (squares), pku70-d cells, Pku70L (triangles), and rad11-D223Y pku70 double mutants, YO002 (circles). For genotypes, see Table 1. Standard deviations are shown by error bars.

Figure 5

Epistasis analysis between rad11-D223Y cells and rad50-d cells for UV sensitivity. The sensitivities to UV light of wild-type cells, JY746 (diamonds), rad11-D223Y cells, YO001 (squares), rad50-d cells, KT120 (triangles) and rad11-D223Y rad50 double mutants, YO003 (circles). For genotypes, see Table 1. Standard deviations are shown by error bars.

Figure 6

rad11⁺ is involved in telomere length maintenance. (A) Schematic presentation of the telomeric and telomere-associated sequences (TAS) of one chromosome arm cloned in the plasmid pNSU70 (47). The positions of telomere and telomere-associated sequences, TAS1, TAS2 and TAS3, are underlined. ApaI-digested telomere sequence was used as a probe for Southern hybridization assays. The chromosome arm is shown by the long gray bar. Restriction enzyme sites are shown above the long gray bar. (B and C) The telomere length of rad11-D223Y, rad11-D223Y pku70-d double mutants and rad11-D223Y rad50-d double mutants was evaluated by Southern hybridization. (B) Lane 1, wild-type cells (JY746); lane 2, rad11-D223Y (YO001); lane 3, pku70-d (PKU70L); lane 4, rad11-D223Y pku70-d double mutants (YO002). (C) Lane 1, wild-type cells (JY746); lane 2, rad11-D223Y (YO001); lane 3, rad50-d (KT120); lane 4, rad11-D223Y rad50-d double mutants (YO003). Telomeres are indicated by arrows. For genotypes, see Table 1. Peaks and distributions of the telomeric DNA-derived bands analyzed using NIH image 1.62 software are shown below. Telomere peaks are indicated by asterisks.

Figure 7

Rad11-Myc is bound to telomere DNA in the ChIP assay. (A) Schematic presentation of the location of the primer set used for the ChIP assay. The position of the primer set used for amplification of telomere DNA is shown above the long gray bar. The positions of telomere and telomere-associated sequences, TAS1, TAS2 and TAS3, are underlined. The chromosome arm is shown by the long gray bar. (B) The ChIP assay of rad11-Myc and rad11-D223Y-Myc. Untagged wild-type cells (JY746), rad11-Myc (YO004) cells and rad11-D223Y-Myc (YO005) cells were used. PCR was performed on whole-cell extracts (WCE) and on chromatin immunoprecipitates (IPs with anti-Myc) using primers to amplify telomere DNA (telomere) and primers to amplify DNA from the eno1⁺ gene (eno1). The relative precipitated fold enrichment is shown underneath each lane. Ratios of telomere signals and eno1⁺ signals were used to calculate relative precipitated fold enrichment.