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. 2006 May 10;5(5):618-28.
doi: 10.1016/j.dnarep.2006.02.005. Epub 2006 Mar 29.

Esc4/Rtt107 and the control of recombination during replication

Jodie K Chin  1 , Vladimir I Bashkirov, Wolf-Dietrich Heyer, Floyd E Romesberg
Affiliations

Esc4/Rtt107 and the control of recombination during replication

Jodie K Chin et al. DNA Repair (Amst). .

Abstract

When replication forks stall during DNA synthesis, cells respond by assembling multi-protein complexes to control the various pathways that stabilize the replication machinery, repair the replication fork, and facilitate the reinitiation of processive DNA synthesis. Increasing evidence suggests that cells have evolved scaffolding proteins to orchestrate and control the assembly of these repair complexes, typified in mammalian cells by several BRCT-motif containing proteins, such as Brca1, Xrcc1, and 53BP1. In Saccharomyces cerevisiae, Esc4 contains six such BRCT domains and is required for the most efficient response to a variety of agents that damage DNA. We show that Esc4 interacts with several proteins involved in the repair and processing of stalled or collapsed replication forks, including the recombination protein Rad55. However, the function of Esc4 does not appear to be restricted to a Rad55-dependent process, as we observed an increase in sensitivity to the DNA alkylating agent methane methylsulfonate (MMS) in a esc4Deltarad55Delta mutant, as well as in double mutants of esc4Delta and other recombination genes, compared to the corresponding single mutants. In addition, we show that Esc4 forms multiple nuclear foci in response to treatment with MMS. Similar behavior is also observed in the absence of damage when either of the S-phase checkpoint proteins, Tof1 or Mrc1, is deleted. Thus, we propose that Esc4 associates with ssDNA of stalled forks and acts as a scaffolding protein to recruit and/or modulate the function of other proteins required to reinitiate DNA synthesis.

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Figures

Figure 1
Figure 1
Protein interactions with Esc4 identified by yeast two hybrid screening. (A) Schematic representation of Esc4; BRCT motifs are shown as black boxes. (B) The bait construct expressed by pNLexA-Esc42–660 and the identified interactions. (C) The bait construct expressed by pEG202-Esc4836–1070 and the identified interaction. Regions corresponding to library inserts are shown as hatched boxes and the number of clones isolated for each insert is given in parentheses.
Figure 2
Figure 2
Esc4 co-immunoprecipitates with the Rad55–57 heterodimer. Protein extracts were prepared from control and MMS-treated cells and subjected to immunoprecipitation with anti-Rad57 antibody (lanes 1–4). Experiments lacking the anti-Rad57 antibody were also performed (lanes 5 and 6). Immunoprecipitates were analyzed for the presence of Rad55 and Esc4 with anti-Rad55 and anti-Myc antibodies, respectively. In cells lacking myc-tagged Esc4, only Rad55 is detected (lanes 1 and 2), consistent with the presence of the Rad55–57 heterodimer. In ESC4-13myc cells, both Rad55 and Esc4-13myc are detected (compare lanes 3 and 4 with lanes 5 and 6).
Figure 3
Figure 3
Deletion of ESC4 results in a small increase in the MMS sensitivity of a rad52Δ mutant and a significant increases the MMS sensitivity of rad51Δ, rad54Δ, rad55Δ, and rad59Δ mutants. Ten-fold serial dilutions of logarithmically growing cultures were plated on YPD and YPD containing 0.004% MMS (rad51Δ, rad52Δ, rad54Δ, and rad55Δ) or 0.007% MMS (rad59Δ).
Figure 4
Figure 4
Genetic interactions between ESC4 and MRE11, RAD50, or XRS2. (A) Strains were grown and diluted as described in Figure 3 and plated on YPD and YPD containing 0.0025% MMS. (B) Five tetrads are shown from each of the crosses indicated. Double mutants are indicated by arrows. Tetrads from crosses with wild type (BY4742) and mre11Δ, rad50Δ, and xrs2Δ single mutants yielded four viable spores (data not shown).
Figure 5
Figure 5
Genetic interactions between ESC4 and RAD27, MUS81, MMS4, SLX1, and SLX4. (A–B) Deletion of ESC4 synergistically increases the MMS sensitivity of rad27, mus81Δ, and mms4Δ mutants. Cells were plated as in Figure 3 on 0.003% MMS (rad27), or 0.005% MMS (mus81 and mms4). Deletion of ESC4 and SLX1 or SLX4 is epistatic in response to MMS. (C) Cells were plated as in Figure 3 on 0.018% MMS. (D) Logarithmically growing cells were plated on YPD containing the amount of MMS indicated. The number of colonies appearing on plates lacking MMS was defined as 100% survival. Survival curves represent the average of at least three independent determinations.
Figure 6
Figure 6
Esc4 is phosphorylated in response to MMS. (A) Modification of Esc4 with increasing exposure to 0.1% MMS. An asynchronous culture of strain FR467 was grown to early log phase and treated with MMS; cells were removed at the times indicated and processed as described (see Methods). (B) Cells were grown as in (A), but lysed in buffer without phosphatase inhibitors. Lysate from untreated cells is shown in lane 1. Lysate from cells exposed to 0.1% MMS for 90 minutes is shown in lanes 2 and 3. In lane 3, 22 μg of lysate was treated with 600 units of λ protein phosphatase (New England Biolabs) for 20 min at 30 °C prior to analysis. (C) Phosphorylation of Esc4 is Mec1-dependent. Strains FR672 and FR673 cells were grown as in (A) with or without 0.1% MMS for 90 minutes. (D) Phosphorylation of Esc4 is mildly affected by deletion of RAD53, but not other, downstream, damage checkpoint functions. Strains FR713, FR716, FR718, and FR715 were treated as in (C). (E) Phosphorylation of Esc4 is not dependent on the presence of recombination mediator proteins. Strains FR674, K37-3, K37-7, and K37-15 were treated as in (C). (F) Phosphorylation of Esc4 is not dependent on either Mrc1 or Tof1. Strains FR674, FR1007, and FR1009 were treated as in (C). Arrows on the right side of each panel indicate the different forms of Esc4. Lanes contain 3 μg of total cell lysate, separated on 5% SDS-PAGE.
Figure 7
Figure 7
Esc4 forms S-phase specific foci in response to MMS. (A) Esc4-GFP(S65T) exhibits three types of nuclear localization: diffuse (upper), 1–2 foci at the edge of the nucleus (middle), or ≥3 distinct foci (lower). (B) Treatment of wild-type cells with MMS increases the percentage of budded cells that contain ≥3 foci. (C) Nuclear foci are also observed in S-phase cells in response to treatment with 100 mM HU for 1 hr. (D) Foci observed in the nucleus of undamaged S-phase cells may be localized to the rDNA. rDNA is visualized with Nop1-RFP (red); nucleus is stained with DAPI (blue). In cells lacking either MRC1 (E) or TOF1 (F), a significant number of budded cells contain foci in the absence of MMS.
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