Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

Abstract

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.

Figure 1

S-phase checkpoint response requires Sgs1p but not Top3p. (A) ISA analysis of Rad53p autophosphorylation was performed on GA-1020, GA-1748, GA-1761, GA-1799, GA-1800, GA-2047, GA-2056 and GA-2060 for which the relevant genotype is indicated beside each data set. For each strain, the upper box shows the incorporation of [γ-³²P]ATP into Rad53p, and the bottom panel a Western for RnaseH42 on the same blot (^*). Time (min) after α-factor release is indicated above each panel. std is 5 μl of a sample containing a fixed amount of an HU-activated Rad53p standard that is used to normalise all gels after identical exposure times (see Materials and methods). (B) Quantification of Rad53p autophosphorylation displayed as percentage of std. The quantification shown is an average of two experiments with standard derivations between 5 and 15%. (C) Five-fold serial dilutions for the indicated strains were plated onto YPAD and incubated at 30°C for 3 days. (D) Bud emergence was scored on 100 cells in cultures released into 0.2 M HU after α-factor block. (E) ISA analysis of Rad53p autophosphorylation was performed in GA-2057 bearing either pRS415 (vector), pRS415 with full-length SGS1 (SGS1-FL) or pRS415 expressing helicase-deficient protein (sgs1-hd) on HU, and on random and S-phase cultures without HU. (F) Quantitation of ISA for GA-2057 with vector, SGS1-FL or sgs1-hd as in (E).

Figure 2

Specific interaction between Sgs1p and the Rad53p FHA1 domain. (A) Immunoprecipitation experiment performed with whole-cell extracts (WCEs) from GA-1142 cells (expressing Myc-tagged Sgs1p and HA-tagged Rad53p). WCEs were obtained from either random or HU-blocked cultures and α-HA (12CA5)-coupled Dynabeads were used for co-immunoprecipitation. Blots were probed with α-Myc (9E10) for Sgs1p (upper panel) or α-HA (Rad53, lower panel). (B) Scheme of Sgs1p and Rad53p domains used to fuse to the B42 activator domain in pJG46 or to the lexA DNA binding domain in pGAL-lexA. (C) The two-hybrid assay shows strong interactions between Sgs1-helicase and Rad53-FHA1 (20-fold), and Rad53-FHA2 (12-fold). Point mutations in FHA1 (R70A) or FHA2 (R605A) eliminate this interaction in pull-down assays (data not shown). β-Galactosidase units are described in Materials and methods.

Figure 3

Mrc1p and Sgs1p work on the same pathway for checkpoint activation on HU. (A) Rad53p autophosphorylation was performed as in Figure 1A on isogenic strains GA-2071, GA-2135, GA-2223 and GA-2500. (B) Bar graph showing quantification of Rad53p autophosphorylation as in Figure 1B. (C) Growth curves for the indicated mutants. Cell density (OD₆₀₀) was measured every hour for 7 h. (D) Schematic summary of the data presented in (A).

Figure 4

Sgs1p functions independently of Rad51p to achieve Rad53p induction on HU. (A) Rad53p autophosphorylation induced by 0.2 M HU was performed as in Figure 1A on isogenic strains GA-1762, GA-1911, GA-2049 and GA-2051. The sgs1 rad24 panel from Figure 1A is added to facilitate comparison. (B) Bar graph showing quantification of Rad53p autophosphorylation, as in Figure 1B. (C–F) The viability assays were performed on synchronised cultures released into 0.2 M HU for indicated times, as described in Materials and methods, using the following isogenic strains: GA-1020, GA-1748, GA-1761, GA-1762, GA-1799, GA-1911, GA-2047, GA-2056, GA-2135 and GA-2223.

Figure 5

Top3p and Rad51p are required for DNA pol ɛ stabilisation. (A) Primers that amplify genome regions corresponding to an early-firing origin ARS607 (black bars), a nonorigin site at +4 kb (grey bars) and +14 kb (white bars) as well as a late-firing origin ARS501 (light grey bars) are shown. (B–H) ChIP was performed on Myc-tagged DNA pol ɛ as described in Materials and methods, on synchronised isogenic cultures of GA-2448, GA-2449, GA-2455, GA-2456, GA-2457, GA-2458 and GA-2796. The height of the bars represents the ratios of real-time PCR signals as fold increase of immunoprecipitation over beads alone, based on duplicate runs and multiple independent experiments. Standard deviation is calculated from all experiments.

Figure 6

Sgs1p and Mrc1p act synergistically to stabilise DNA pol ɛ at stalled forks. (A–D) ChIP was performed on Myc-tagged DNA pol ɛ as described in Figure 5 on synchronised isogenic strains GA-2448, GA-2449, GA-2451 and GA-2452. (E) FACS analysis during S-phase progression in the absence of damage for GA-1020, GA-2135 and GA-2223.

Figure 7

Different pathways use the RecQ helicase in response to replication stalling. Stalled replication forks activate Rad53p kinase in the absence of strand breaks in an Sgs1p-, Mrc1- and Mec1-dependent pathway that is independent of Top3p and Rad51p. Forks are stabilised by two mechanisms, including one that requires Rad51p and the Sgs1p/Top3p complex. We proposed that the Rad51p-dependent pathway leads to the formation of a four-way DNA junction due to fork reversal. DSBs will form at some of the stalled replication forks, and will activate the intra-S damage pathway (breakage pathway) for checkpoint activation, which relies on Rad24p and the 9-1-1 complex.