The PCNA unloader Elg1 promotes recombination at collapsed replication forks in fission yeast
- University of Oxford, United Kingdom
Figures
Figure 1 with 1 supplement
Spontaneous (RTS1-IO) and RTS1-AO-induced direct repeat recombination in wild-type and elg1∆ strains.
(A) Map showing the position of RTS1, origins of replication and sites of insertion for the direct repeat recombination reporter on chromosome 3. (B) Schematic of the direct repeat recombination …
Figure 1—figure supplement 1
Elg1 is not required for replication fork blocking at RTS1-AO.
(A) Schematic showing the EcoNI restriction fragment and probe used for the 2DGE analysis in B. (B) 2DGE of replication intermediates in the EcoNI fragment shown in A. The DNA was extracted from …
Figure 2
Comparison of CFP-PCNA fluorescence in wild-type and elg1∆ strains.
(A) Stills taken from four separate time-lapse movies (two each for wild-type and elg1∆) showing the different stages of nuclear CFP-PCNA fluorescence at 5 min intervals during representative cell …
Figure 3
Spontaneous (RTS1-IO) and RTS1-AO-induced direct repeat recombination in pcn1+ and pcn1D150E strains with and without elg1.
(A) Ade+ recombinant frequencies for strains MCW9394, MCW9390, MCW9183 and MCW9187. (B) Ade+ recombinant frequencies for strains MCW9396, MCW9392, MCW9185 and MCW9189. Data are mean values with …
Figure 4 with 1 supplement
Effect of deleting srs2 and fbh1 on the frequency of RTS1-AO-induced direct repeat recombination in wild-type and elg1∆ strains.
The strains are MCW4713, MCW7708, FO1750, MCW8330, FO1816 and MCW8946. Data are mean values with error bars showing 1 SD. Significant differences relative to equivalent elg1+ strain values are …
Figure 4—figure supplement 1
Growth and genotoxin sensitivities of elg1∆, srs2∆ and fbh1∆ single and double mutants.
(A) Spot assay comparing the growth, and CPT and MMS sensitivities of wild-type (MCW1221), elg1∆ (MCW7586), srs2∆ (MCW1017) and srs2∆ elg1∆ (MCW8332) strains. (B) Spot assay comparing the growth, …
Figure 5
CFP-Rad51 and Rad52-YFP foci colocalization with RTS1-AO in wild-type and elg1∆ strains.
(A) Schematic showing key components of the strains used for imaging, including the modification of the 0 kb site recombination reporter with a 115 repeat lacO array. (B) Examples of two wild-type …
Figure 6
Elg1 promotes IFSA in the presence and absence of Rad51.
(A) Schematic showing the insertion of the 5 kb DNA spacer at the 0 kb site recombination reporter. (B) Ade+ recombinant frequencies for strains MCW8023, MCW8941, MCW8136 and MCW8943. Data are mean …
Figure 7
Model showing how Elg1 promotes recombination at a blocked replication fork.
Upon encounter with RTS1-AO, the replication fork undergoes reversal forming a ‘chicken foot’ structure with an exposed duplex DNA end. Resection of this DNA duplex, by the nucleolytic activity of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (S. pombe) | various strains | Ahn et al., 2005 | standard laboratory strain (972) derivatives; see Supplementary file 2 | |
| Strain, strain background (S. pombe) | various strains | Nguyen et al., 2015 | standard laboratory strain (972) derivatives; see Supplementary file 2 | |
| Strain, strain background (S. pombe) | various strains | this paper | standard laboratory strain (972) derivatives; see Supplementary file 2 | |
| Strain, strain background (S. pombe) | various strains | Lorenz et al., 2009 | standard laboratory strain (972) derivatives; see Supplementary file 2 | |
| Strain, strain background (S. pombe) | various strains | Morrow et al., 2017 | standard laboratory strain (972) derivatives; see Supplementary file 2 | |
| Recombinant DNA reagent | pMW777 | this paper | plasmid; see Materials and methods | |
| Recombinant DNA reagent | pST2 | this paper | plasmid; see Materials and methods | |
| Sequence-based reagent | oMW1884 | this paper | oligonucleotide; see Materials and methods | |
| Sequence-based reagent | oMW1885 | this paper | oligonucleotide; see Materials and methods |
Additional files
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Supplementary file 1
Direct repeat recombinant frequencies.
- https://doi.org/10.7554/eLife.47277.011
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Supplementary file 2
Schizosaccharomyces pombe strains.
- https://doi.org/10.7554/eLife.47277.012
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Transparent reporting form
- https://doi.org/10.7554/eLife.47277.013
