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. 2016 Sep 30;44(17):8199-215.
doi: 10.1093/nar/gkw535. Epub 2016 Jun 13.

The Shu Complex Promotes Error-Free Tolerance of Alkylation-Induced Base Excision Repair Products

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Free PMC article

The Shu Complex Promotes Error-Free Tolerance of Alkylation-Induced Base Excision Repair Products

Stephen K Godin et al. Nucleic Acids Res. .
Free PMC article

Abstract

Here, we investigate the role of the budding yeast Shu complex in promoting homologous recombination (HR) upon replication fork damage. We recently found that the Shu complex stimulates Rad51 filament formation during HR through its physical interactions with Rad55-Rad57. Unlike other HR factors, Shu complex mutants are primarily sensitive to replicative stress caused by MMS and not to more direct DNA breaks. Here, we uncover a novel role for the Shu complex in the repair of specific MMS-induced DNA lesions and elucidate the interplay between HR and translesion DNA synthesis. We find that the Shu complex promotes high-fidelity bypass of MMS-induced alkylation damage, such as N3-methyladenine, as well as bypassing the abasic sites generated after Mag1 removes N3-methyladenine lesions. Furthermore, we find that the Shu complex responds to ssDNA breaks generated in cells lacking the abasic site endonucleases. At each lesion, the Shu complex promotes Rad51-dependent HR as the primary repair/tolerance mechanism over error-prone translesion DNA polymerases. Together, our work demonstrates that the Shu complex's promotion of Rad51 pre-synaptic filaments is critical for high-fidelity bypass of multiple replication-blocking lesion.

Figures

Figure 1.
Figure 1.
Schematic of base excision repair (BER) and the Shu complex primary sensitivity to methylmethane sulfonate (MMS) induced DNA damage. (A) Cartoon of BER pathway adapted from (51). The black arrows indicate the major pathway. (B) The indicated strains were 5-fold serially diluted onto YPD medium, YPD medium containing 0.006% or 0.02% MMS, SC medium or SC medium containing 40 μg/ml cisplatin. The plates were incubated for two days at 30°C and photographed. (C) Same as (B) except that the YPD plate was exposed to 70 Gray ionizing radiation (IR) or 50 J/m2 ultra-violet (UV), or contained 100 mM hydroxyurea (HU) or 3 mM H2O2. (D) Same as (B) except that 2 mM etoposide was added to the YPD medium. (E) Same as (B) except that 50 μg/ml CPT was added to the YPD medium.
Figure 2.
Figure 2.
In the absence of the DNA glycosylase MAG1, Shu complex disrupted cells are hypersensitive to MMS-induced alkylation damage. (A) Cartoon demonstrating what DNA processing step is blocked by mag1Δ disruption. (B) The indicated yeast strains were 5-fold serially diluted onto YPD medium or YPD medium containing MMS (0.002%, 0.006%, 0.012% and 0.02%) and incubated for two days at 30°C and subsequently photographed. (C) Mutation frequencies of WT, csm2Δ, mag1Δ and csm2Δ mag1Δ cells were determined at the CAN1 locus both spontaneously and upon MMS exposure. Three experiments with five separate isolates were analyzed and averaged. The graph represents the average of the medians with standard deviations plotted. Significance was determined by t-test. (D) Schematic of a direct-repeat recombination assay, where Rad51-dependent gene conversion (GC; URA3+ LEU2+) and Rad51-independent single-strand annealing (SSA; URA3- LEU2+) repair can be measured. Total recombination rates of the indicated strains are shown before and after MMS treatment. (E) The levels of Rad51-dependent (GC) and Rad51-independent (SSA) repair for each condition shown in (D). For (D) and (E), each condition was repeated 3 to 5 times and the average recombination rates were calculated as described in the methods section, are graphed.
Figure 3.
Figure 3.
CSM2 disrupted cells are sensitive to AP sites and/or 3′-dRPs as demonstrated by the synthetic sensitivity of csm2Δ with apn1Δ apn2Δ on MMS containing medium. (A) Cartoon demonstrating what DNA processing steps are blocked by apn1Δ, apn2Δ disruption and accumulation of AP sites and 3′-dRPs. (B and C) The indicated yeast strains were 5-fold serially diluted onto rich medium (YPD) or YPD medium containing the indicated dose of MMS. The plates were incubated at 30°C for two days and photographed. (D) In CSM2 disrupted cells, Mag1 is epistatic to Apn1 and Apn2. The indicated yeast strains were 5-fold serially diluted onto YPD or YPD with MMS as described for (B and C). (E) WT, apn1Δ apn2Δ, csm2Δ and apn1Δ apn2Δ csm2Δ cells expressing Rad52-YFP and Rfa1-CFP were analyzed by fluorescent microscopy for Rad52 and Rfa1 focus formation (indicated by a white arrow). A single z-plane is shown for WT and apn1Δ apn2Δ csm2Δ cells. (F) The percentage of WT, apn1Δ apn2Δ, csm2Δ and apn1Δ apn2Δ csm2Δ S/G2/M cells exhibiting a spontaneous nuclear Rad52-YFP or Rfa1-CFP focus. Three individual experiments totaling 300–400 cells were analyzed per genotype and standard error of the means plotted. (G) The cell cycle distribution of the indicated genotypes in (E) were measured as a percentage of G1 cells (unbudded) and S/G2/M cells (budded) from the images acquired in (E).
Figure 4.
Figure 4.
CSM2 is important for repair of MMS-induced damage during S phase but not G2 phase. (A) Protein levels of Csm2, Clb2 (S/G2-phase control) and Kar2 (loading control) in asynchronous (ASN) cells or after release from G1 arrest by alpha factor. (B) Chromosome shattering and restitution in WT, apn1Δ apn2Δ and apn1Δ apn2Δ csm2Δ cells arrested in G2 cell cycle by nocodazole. Chromosomes are harvested at the indicated time points before and after treatment with MMS and separated by PFGE as described in the methods. (C) Chromosome restitution following progression through S phase in WT or csm2Δ cells treated with MMS.
Figure 5.
Figure 5.
CSM2 disrupted cells are sensitive to AP sites as demonstrated by the synthetic sensitivity of csm2Δ with apn1Δ apn2Δ ntg1Δ ntg2Δ on MMS containing medium. (A) Cartoon demonstrating what DNA processing steps are blocked by apn1Δ, apn2Δ, ntg1Δ and ntg2Δ disruption and accumulation of AP sites. (B) The indicated yeast strains were 5-fold serially diluted onto rich medium (YPD) or YPD medium containing the indicated dose of MMS. The plates were incubated at 30°C for two days and photographed. (C) The total recombination rates, as in Figure 2D. (D) The levels of Rad51-dependent and independent repair as in Figure 2E.
Figure 6.
Figure 6.
Cells containing csm2-F46A are defective for tolerance of BER lesions. (A–C) The indicated yeast strains were 5-fold serially diluted onto rich medium (YPD) or YPD medium containing the indicated doses of MMS.
Figure 7.
Figure 7.
Expression of human Polβ partially suppresses the MMS sensitivity of rad27Δ cells and the synthetic growth defect observed in csm2Δ rad27Δ double mutant cells. (A) Cartoon indicating what processing step is blocked by rad27Δ disruption. (B) The indicated yeast strains were 5-fold serially diluted onto YPD medium or YPD medium containing 0.002%, 0.006%, 0.012% or 0.02% MMS where indicated. The plates were incubated at 30°C for two days and photographed. (C) WT cells or rad27Δ cells which also express either an empty vector (pAG414-GAL) or the human Polβ in an expression plasmid (pAG414-GAL-Polβ) were 5-fold serially diluted and plated onto YPRaffinose with galactose (YPR + GAL) medium or YPR + GAL medium containing 0.02% MMS. The cells were incubated at 30°C for two days and photographed. (D) WT, shu1Δ, shu2Δ, psy3Δ, csm2Δ cells were transformed with pAG414-GAL-Polβ, 5-fold serially diluted and plated onto YPR + GAL medium or YPR + GAL medium containing 0.02% MMS. The plates were incubated at 30°C for two days and photographed. (E) WT, rad27Δ, csm2Δ, csm2Δ rad27Δ cells were transformed with either the pAG414-GPD (Empty Vector) or the pAG414-GPD-Polβ (Polβ) plasmid and 5-fold serially diluted onto YPD or YPD medium containing 0.002% or 0.006% MMS. The plates were incubated at 30°C for two days and photographed.
Figure 8.
Figure 8.
The translesion DNA polymerase ζ is necessary for lesion bypass in MMS-exposed csm2Δ apn1Δ apn2Δ or csm2Δ mag1Δ cells. (A–C) The indicated genotypes were 5-fold serially diluted onto YPD medium or YPD medium containing 0.002%, 0.006%, 0.012% or 0.02% MMS. The plates were incubated at 30°C for two days and photographed.
Figure 9.
Figure 9.
Analysis of CSM2-disrupted cells with error-free PRR disruption (rad5Δ, ubc13Δ, mms2Δ) by MMS sensitivity, mutation frequencies and direct repeat recombination rates. (A) The indicated yeast strains were 5-fold serially diluted onto rich medium (YPD) and YPD medium containing the indicated dose of MMS. The plates were incubated for two days at 30°C and photographed. (B) Mutation frequencies were determined in the indicated strains at the CAN1 locus. Three experiments with five separate isolates were analyzed and averaged. The graph represents the average of the medians with standard deviations plotted. Significance was determined by t-test. (C) The rate of gene conversion and SSA-like events were measured as described in Figure 2E. Three to four experiments were done per strain and standard deviations plotted. Significance was determined by t-test.
Figure 10.
Figure 10.
Overview repair model of MMS-induced DNA damage in budding yeast by BER, translesion DNA synthesis (TLS) and HR (Adapted from (55)). MMS damage leads to base lesions excised by the DNA glycosylase Mag1 to create an AP site. Both the MMS-induced base lesions and AP sites can lead to a replication fork block, which can either be bypassed by the TLS pathway (requiring Rev3) resulting in increased mutation frequency and therefore, error-prone repair (69). During BER, Apn1/Apn2 further processes the AP site leading to 5′-dRPs, which are filled in by DNA polymerase ϵ and the 5′-dRP flap is cleaved by Rad27. Lig1 ligates the resulting nick. The 5′-dRP and flap as well as the nicked DNA can lead to replication fork collapse and DSB formation. The resulting DSB can be repaired by the high-fidelity HR pathway via the Shu complex, which is important for the critical Rad51 filament formation step of HR.

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