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. 2006 Jan;17(1):539-48.
doi: 10.1091/mbc.e05-08-0785. Epub 2005 Nov 2.

Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4

Tania M Roberts  1 , Michael S Kobor, Suzanne A Bastin-Shanower, Miki Ii, Sonja A Horte, Jennifer W Gin, Andrew Emili, Jasper Rine, Steven J Brill, Grant W Brown
Affiliations

Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4

Tania M Roberts et al. Mol Biol Cell. 2006 Jan.

Abstract

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal-domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint protein kinase Mec1, although the mechanism by which Rtt107 is targeted by Mec1 after checkpoint activation is currently unclear. Slx4, a component of the Slx1-Slx4 structure-specific nuclease, formed a complex with Rtt107. Deletion of SLX4 conferred many of the same DNA-repair defects observed in rtt107delta, including DNA damage sensitivity, prolonged DNA damage checkpoint activation, and increased spontaneous DNA damage. These phenotypes were not shared by the Slx4 binding partner Slx1, suggesting that the functions of the Slx4 and Slx1 proteins in the DNA damage response were not identical. Of particular interest, Slx4, but not Slx1, was required for phosphorylation of Rtt107 by Mec1 in vivo, indicating that Slx4 was a mediator of DNA damage-dependent phosphorylation of the checkpoint effector Rtt107. We propose that Slx4 has roles in the DNA damage response that are distinct from the function of Slx1-Slx4 in maintaining rDNA structure and that Slx4-dependent phosphorylation of Rtt107 by Mec1 is critical for replication restart after alkylation damage.

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Figures

Figure 1.
Figure 1.
Rtt107 physically interacts with Slx4 and Slx1. (A) Extracts from yeast strains expressing the indicated epitope-tagged proteins were immunoprecipitated with IgG agarose or α-VSV followed by protein G agarose. Ten percent of the input extract (E) and the entire immunoprecipitate (IP) were fractionated by SDS-PAGE. Immunoblots were probed with α-VSV to detect Rtt107-VSV, peroxidase-anti-peroxidase to detect Slx4-TAP. (B) Extracts were treated as in A, and immunoblots were probed with α-HA to detect Slx4-HA and Slx1-HA or rabbit IgG to detect Rtt107-TAP.
Figure 2.
Figure 2.
The Rtt107-Slx4 interaction is direct and is independent of Slx1, DNA damage, and DNA damage checkpoints. (A and B) Logarithmically growing cultures were treated with 0 or 0.03% MMS for 1 h. Extracts from yeast strains expressing the indicated epitope-tagged proteins were immunoprecipitated with IgG agarose. The immunoprecipitate was fractionated by SDS-PAGE, and immunoblots were probed with α-VSV to detect Rtt107-VSV and peroxidase-anti-peroxidase to detect Slx4-TAP. (C) Recombinant Rtt107, Slx1, and His6-Slx4 were coexpressed in E. coli, and the His6-Slx4 and associated proteins were purified and resolved on an SDS-polyacrylamide gel. The gel was stained with silver. Note that Rtt107 and His6-Slx4 have very similar mobility in this gel system. (D) Recombinant Slx4 and His6-RTT107-FLAG were coexpressed in E. coli and the His6-Rtt107-FLAG was purified. Purified proteins were fractionated on SDS-PAGE and stained with Coomassie blue. Note the slower mobility of the His6-Rtt107-FLAG compared with His6-Rtt107 and the faster mobility of Slx4 compared with His6-Slx4. (E) Truncated and full-length Rtt107-TAP were coexpressed with Slx4-FLAG. Rtt107-TAP and associated proteins were immunoprecipitated with rabbit IgG. Immunoprecipitates were fractionated by SDS-PAGE, and proteins were detected by probing the immunoblot with α-FLAG to detect Slx4-FLAG or rabbit IgG to detect Rtt107-TAP, Rtt107(1-511)-TAP, and Rtt107(512-1070)-TAP. The vector lane is a control strain in which no Rtt107-TAP is present.
Figure 3.
Figure 3.
Rtt107 and Slx4 are important for the recovery from MMS-induced DNA damage. (A) Cells were α-factor blocked in G1 and released into media containing 0.03% MMS for 1 h. Samples were taken at the indicated times, samples were fixed with TCA, and extracts were fractionated on SDS-PAGE for in situ kinase assay of Rad53. (B) Levels of Rad53 activity after treatment with MMS, relative to Rad53 activity in the asynchronous undamaged sample (ASY) are plotted. Average of two independent experiments is shown. (C) Chromosomes plugs were prepared from untreated cells, from cells treated with MMS, and from cells recovering from MMS damage. Chromosomes were separated by CHEF gel electrophoresis and detected by ethidium bromide staining.
Figure 4.
Figure 4.
rtt107Δ and slx4Δ mutants accumulate in anaphase during recovery from MMS-induced damage. (A) Logarithmically growing cultures were treated with 0.03% MMS for 1 h, samples were withdrawn at the indicated times, and stained with DAPI to examine nuclear morphology; differential interference contrast images (left), and corresponding DAPI images (right) are shown. (B and C) Cells treated as in A were sampled at the indicated times. The percentage of cells with a large bud (% G2/M) and percentage of cells with a large bud and an elongated nucleus spanning the bud neck (% elongated nucleus) are plotted.
Figure 5.
Figure 5.
Rtt107 and Slx4 suppress spontaneous DNA damage. (A) Logarithmically growing cells expressing Ddc2-YFP were visualized by fluorescence microscopy; representative fields are shown. (B) The percentage of cells with Ddc2-YFP foci in G1 (unbudded) and G2/S/M (budded) cells is plotted for the indicated strains.
Figure 6.
Figure 6.
Slx4 is required for Mec1 phosphorylation of Rtt107 in response to MMS induced damage. (A) Logarithmically growing cultures were treated with 0 or 0.03% MMS for 1 h. Samples were fixed with TCA, extracts were fractionated on SDS-PAGE, and immunoblots were probed for Rtt107-VSV. (B) Rtt107-VSV was immunoprecipitated from yeast extracts of the indicated strains, and the immunoprecipitates were fractionated by SDS-PAGE. The corresponding immunoblot was probed with α-phospho-[S/T]Q to detect phosphorylated Mec1 consensus sites, or with α-VSV to detect Rtt107-VSV.
Figure 7.
Figure 7.
RTT107 and SLX4 make independent contributions to DNA damage resistance. (A) Ten-fold serial dilutions of cells were spotted onto YPD or YPD containing 0.03% MMS and incubated at 30°C for 3 d. (B) Logarithmically growing cultures were blocked in G1 with α-factor and released into media containing 0.04% MMS. Samples were withdrawn at the indicated times and plated on YPD to determine cell viability. The percentage of viable cells relative to the number of viable cells at t = 0 is shown. The average of three independent experiments is plotted.
Figure 8.
Figure 8.
Model of the role of Rtt107 and Slx4 in the DNA damage response.
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