Figure 1.
Schematic representation of 2D gel replication intermediates and genomic maps.
(A) The genomic region containing the ARS305 origin and the flanking regions on chromosome III. E and H stand for EcoRV and HindIII, respectively. N stands for NcoI. The ARS305 probe spans from 39026 to 41647, the ARS301 probe from 10135 to 11416. (B) Schematic representation of the replication intermediates visualized by 2D gel electrophoresis.
Figure 2.
Rad55, but not Rad55 phosphorylation by Rad53 or Rad59, is required for template switch replication.
(A) sgs1Δ (HY1465), sgs1Δ rad55Δ (HY1460) and sgs1Δ rad55-S2,8,14A (HY0799) and (B) sgs1Δ (FY1058) and sgs1Δ rad59Δ (HY1414) were arrested in G1 with α-factor (A) or with nocodazole in G2 (B) and released in medium containing MMS 0.033% at 30°C. At the indicated time-points samples were taken, the genomic DNA was extracted and digested with EcoRV and HindIII and the replication intermediates were analyzed by 2D gel with a probe recognizing the ARS305 region.
Figure 3.
RPA, promoting the strand invasion step of homologous recombination, is required for template switch replication.
(A) sgs1 (HY1461) and sgs1 rfa1-t11 (HY1459) were synchronized in G2 with nocodazole and released in medium containing MMS 0.033% at 28°C. The replication intermediates were digested with NcoI and analyzed with the ARS305 probe. (B) Exponentially growing wild-type (W303-1A) and rfa1-t11 (HY1464) cells were treated for 2 and 4 hours with MMS 0.02%. Western blot analysis was performed to detect Rad53 phosphorylation.
Figure 4.
Exo1 contributes to damage-bypass replication by template switch.
2D gel analysis of replication intermediates digested with NcoI from sgs1 (HY1461) and sgs1 exo1Δ (HY1448) cells synchronized with nocodazole and released in medium containing MMS 0.033% at 28°C.
Figure 5.
Translesion synthesis polymerases do not contribute to the DNA synthesis step of template switch.
(A) sgs1Δ (HY1465) and sgs1Δ rad30Δ (HY1467) cells and (B) sgs1Δ (HY1465) and sgs1Δ rev7Δ rev1Δ rad30Δ (HY1468) cells were synchronized in G2 with nocodazole and then released in YPD medium containing MMS 0.033%. Both experiments were performed at 28°C and samples were taken for 2D gel analysis; the DNA was digested with EcoRV and HindIII and the membrane hybridized with a probe corresponding to ARS305.
Figure 6.
Polδ but not Polε, is required for template switch replication.
2D gel analysis of replication intermediates in (A) sgs1 (HY1455), sgs1 pol2-11 (HY1456) and (B) sgs1 (HY0100), sgs1 cdc2-1 (HY0103). The experiments were performed at the semi-permissive temperature of 30°C. The DNA samples were digested with HindIII and EcoRV and the membranes hybridized with a probe corresponding to ARS305.
Figure 7.
The effect of the replication-proficient pol3-ct mutation in template switch replication.
2D gel analysis of replication intermediates forming at ARS305 from sgs1 (HY1461) and sgs1 pol3-ct (HY1257) cells replicating in the presence of MMS damage at the permissive temperature of 30°C. The p-values obtained from unpaired t-tests for 120 min (P = 0. 0089) and 180 min (P = 0. 0022) indicate that the differences between sgs1 and sgs1 pol3-ct are statistically significant.
Figure 8.
The effect of the pol32Δ mutation in template switch replication.
2D gel analysis of replication intermediates from GAL-SGS1 (FY1359) and GAL-SGS1 pol32Δ (FY1379) under conditions in which SGS1 expression was shut-down by the addition of glucose. The cells were grown to log phase in YP-media containing raffinose and galactose at 30°C, arrested in G1 and then released in YPD containing MMS 0.033% at 25°C for 4 hours. The DNA samples were digested with HindIII and EcoRV and the membranes hybridized with a probe corresponding to ARS305.
Figure 9.
Model for factors contributing to template switch replication.
DNA damage during replication can cause uncoupling between the leading and lagging strands. In this model the DNA lesion is represented on the leading strand behind the replication fork. The gaps are further processed by the Exo1 nuclease to expose ssDNA. RPA coats ssDNA to stimulate recombination events. Additional recombination factors including Rad52, Rad51, and Rad55-Rad57 promote strand invasion into the homologous duplex. The newly synthesized sister chromatid serves as a template for DNA synthesis, which is mediated mainly by Polδ. The activities regulating Polδ-loading and Polδ-mediated DNA repair synthesis remain to be investigated; they may involve Rad18-Rad5-Mms2 functions and PCNA modifications. The 3′end of the nascent invading strand returns to its parental template, giving rise to the X-shaped template switch intermediates. The possibility that Rad51 stabilizes the ssDNA stretches of the X-intermediates into paranemic junctions is also represented. The template switch intermediates are then resolved by the action of Sgs1-Top3.