Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs
© Zappulla et al; licensee BioMed Central Ltd. 2006
Received: 30 August 2006
Accepted: 09 November 2006
Published: 09 November 2006
By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to e stablish s ilent c hromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions.
Targeted silencing by Rtt107/Esc4 was dependent on the SIR genes, which encode obligatory structural and enzymatic components of yeast silent chromatin. Based on its sequence, Rtt107/Esc4 was predicted to contain six BRCT motifs. This motif, originally identified in the human breast tumor suppressor gene BRCA1, is a protein interaction domain. The targeted silencing activity of Rtt107/Esc4 resided within the C-terminal two BRCT motifs, and this region of the protein bound to Sir3 in two-hybrid tests. Deletion of RTT107/ESC4 caused sensitivity to the DNA damaging agent MMS as well as to hydroxyurea. A two-hybrid screen showed that the N-terminal BRCT motifs of Rtt107/Esc4 bound to Slx4, a protein previously shown to be involved in DNA repair and required for viability in a strain lacking the DNA helicase Sgs1. Like SLX genes, RTT107ESC4 interacted genetically with SGS1; esc4 Δ sgs1 Δ mutants were viable, but exhibited a slow-growth phenotype and also a synergistic DNA repair defect.
Rtt107/Esc4 binds to the silencing protein Sir3 and the DNA repair protein Slx4 via different BRCT motifs, thus providing a bridge linking silent chromatin to DNA repair enzymes.
Transcriptional silencing in the budding yeast Saccharomyces cerevisiae occurs at the silent mating-type loci HMR and HML, telomeres, and at the rDNA locus. At all of these silenced regions, DNA binding proteins recognize specific motifs and recruit a silencing protein complex (reviewed in ). HMR and HML are flanked by E and I "silencers." Each silencer has binding sites for ORC, and Rap1 or Abf1. The potent HMR-E silencer has a binding site for all three proteins. At telomeres, Rap1 also contributes a critical DNA-binding function, binding to the TG1–3 repeats.
At both the silent mating loci and at telomeres, the DNA-binding proteins recruit a Sir protein complex that can spread to silence genes at a distance (reviewed in ). At HMR-E, for example, this is achieved by ORC recruitment of Sir1 via a Sir1-Orc1 interaction , and Rap1 and Abf1 binding to Sir4 and Sir3 [4, 5]. Sir4 and Sir3 multimerize, both with themselves and each other [4, 6]. Sir4 also binds Sir2, and Sir2 plays a crucial role in the spreading of a Sir2, Sir3, Sir4 complex on chromatin by deacetylating histone H4 lysine 16. The deacetylation produces a novel product, 2' O-acetyl-ADP-ribose [7–9]. This compound has recently been shown to produce a conformational change in Sir3 that is likely to promote spreading of the Sir complex. Sir2, Sir3 and Sir4 are essential for silencing at the HM loci and at telomeres, while Sir1 plays a prominent role in silencing at the mating type loci but not at telomeres. The Yku70/Yku80 heterodimer that binds to DNA ends plays an important role in silencing at telomeres, while being dispensable for silencing at HM loci .
In this report we show that Esc4 has 6 BRCT motifs and that they are important for its function. Many proteins that function in repair, and even a silencing protein, Rap1, contain BRCT (BR CA1 C-terminus) motifs. This motif was first identified by database searching using the C-terminus of the human breast cancer susceptibility protein, BRCA1 . Since then, BRCT motifs from the human DNA repair protein XRCC1 and the two tandem BRCT motifs of BRCA1 protein have been crystallized [24, 25] and crystal and/or solution structures have also been solved of BRCTs from 53BP1, DNA ligase III, and an NAD-dependent DNA ligase [26–29]. More recently, it was discovered that BRCT motifs could specifically bind to phosphoserine-containing proteins [30, 31] and structures of such complexes were subsequently also determined [32–34]. Overall, BRCT motifs are thought to mediate a diverse array of protein-protein interactions, binding to proteins with different structures, as well as to other BRCTs, both inter- and intra-molecularly (reviewed in ).
DNA repair requires many DNA-modifying enzymes such as nucleases, ligases, topoisomerases, polymerases and helicases. Saccharomyces cerevisiae SGS1 is a member of the RecQ family of genes encoding DNA helicases. In addition to Escherichia coli recQ, this helicase gene family includes human BLM, WRN, RECQL, and Schizosaccharomyces pombe rqh1+. Members of the RecQ family of helicases have been implicated in genomic stability, aging and cancer. Yeast SGS1 has been shown by several labs to suppress DNA recombination, gross chromosomal rearrangements, and Ty1 transposition, and to exhibit 3' to 5' helicase activity [20, 36–38]. Six SLX genes have been isolated in a synthetic lethal screen using an sgs1 Δ mutant . Two of these genes, SLX2 and SLX3, encode the Mus81/Mms4 nuclease that acts on branched DNA structures. SLX1 is also predicted to be a nuclease, based on its sequence . Slx1 and Slx4 coimmunoprecipitate and slx1 and slx4 mutants display similar phenotypes and have been proposed to function together . In addition, a genome-wide genetic interaction (synthetic genetic array, SGA) screen isolated a total of 24 genes that show a synthetic growth interaction with an sgs1 Δ mutation . This screen identified 4 of the 6 SLX genes previously identified in the sgs1 Δ synthetic lethal screen described above , as well as three other genes known to show synthetic interactions [39, 42–45]. The screen also identified 16 other genes that caused synthetic lethality or sickness when mutated with sgs1 Δ. One of these genes was ESC4. Subsequently, there have been other reports confirming this genetic interaction between SGS1 and ESC4 [46, 47].
Here we demonstrate that the C-terminal two BRCT motifs of Esc4 bind to Sir3 and are sufficient for SIR-dependent targeted silencing at HMR. Furthermore, the N-terminal four BRCT motifs in Esc4 bind to Slx4, thus linking this DNA repair protein to silent chromatin.
Esc4 establishes targeted silencing when targeted to HMR
In a screen described previously, we identified Esc4 as a protein that could restore silencing when targeted to an HMR locus harboring a deletion of the HMR-E silencer . Targeting of proteins to HMR was mediated by the binding of a Gal4 DNA binding domain (GBD)-hybrid protein to a Gal4 DNA binding site (G) that replaced the HMR-E silencer (Figure 1). Silencing was assessed using a URA3 reporter gene integrated at the HMR locus.
Targeted silencing by Esc4 is SIR-dependent
To test whether targeted silencing by GBD-Esc4 was SIR-dependent, it was tested in strains deleted for the SIR2, SIR3 or SIR4 genes. As seen in Figure 2, silencing by Esc4 required each of these SIR genes. This was expected since targeted silencing by other proteins has been shown to be SIR-dependent in every case examined previously [14, 16]. It thus seemed likely that Esc4 caused silencing by recruiting the Sir protein complex.
Saccharomyces Esc4 proteins contain six BRCT motifs and are homologous to S. pombe Brc1
The six BRCT motifs of Esc4 protein exist as a set of four tandem motifs at the N-terminus of the protein and two more at the C-terminus (Figure 3, yellow lines). These two sets of BRCT motifs are separated by a 375 amino acid linker region. Although there are proteins from various budding yeasts with compelling sequence similarity to S. cerevisiae Esc4 (e.g. Ashbya gossipii, Kluyveromyces lactis, and Candida glabrata, as shown in Figure 3), currently the only obvious non-budding yeast homolog candidate is Brc1 from the evolutionarily distant fission yeast Schizosaccharomyces pombe (Figure 3, bottom line). S. pombe Brc1 is 878 amino acids in length and also contains four BRCTs at the N-terminus and two at the C-terminus, separated by a linker region. Esc4 and Brc1 are 52% similar and 21% identical overall. Human and mouse PTIP proteins have been reported to be quite similar in sequence [21, 53], but do not contain the classically-conserved C-terminal aromatic residue present in all of the six BRCTs highlighted with a red asterisk in the alignment shown in Figure 3. Therefore, it is not yet clear if these proteins are bona fide Esc4/Brc1 homologs.
The C-terminal two BRCT motifs of Esc4 are sufficient for targeted silencing
Yeast strains used in this study
Reference or Source
MAT a leu2–3,112 ura3-1 his3–11,15 trp1–1 ade2-1 can1–100
MAT α version of W303-1a
Diploid from W303-1a × W303-1b
W303 MAT a /MAT α esc4Δ::his5+/ESC4
W303 MAT a /MAT α esc4Δ::his5+/esc4Δ::his5+
W303-1a esc4 Δ::his5+
W303-1b esc4 Δ::his5+
W303-1b hmr::URA3 HMR-E (aeb::G) gal4::LEU2
YEA78 but HMR-E (Aeb::G)
YEA78 but HMR-E (aeB::G)
YEA76 sir2 Δ::kanMX6
YEA76 but GAL4 sir3::LEU2
YEA76 sir4 Δ::kanMX6
W303-1b adh4::URA3-(C1–3A) n
YDS631 esc4 Δ::his5+
W303-1a slx4 Δ::his5+
W303-1b slx4 Δ::his5+
W303-1a slx4 Δ::kanMX6
W303-1b slx4 Δ::kanMX6
W303-1b sgs1 Δ::kanMX6
W303-1_ sgs1 Δ::kanMX6 esc4 Δ::his5+
W303-1a esc4 Δ::his5+slx4 Δ::kanMX6
W303-1a asf1 Δ::his5+
MAT a his3 Δ200 trp1–901 leu2–3,112 ade2 URA3::(lexAop)8-lacZ LYS2::(lexAop)4-HIS3
L40 sir3 Δ::kanMX6
L40 sir4 Δ::kanMX6
The C-terminal BRCT motifs of Esc4 interact with Sir3
Esc4 binds to Sir3 in the two-hybrid system
Interaction in two-hybrid strainsa
L40 Δ sir3
L40 Δ sir4
The N-terminal four BRCT motifs of Esc4 bind to Slx4
The amino-terminal BRCT motifs of Esc4 bind to Slx4
Number of isolates
Genetic and phenotypic analysis of ESC4
A heterozygous diploid strain with a complete deletion of ESC4 was constructed and dissected to generate a null mutant haploid. This esc4 Δ mutant grew normally and also mated with normal efficiency (suggesting no gross defect in HM silencing). Furthermore, when an esc4 Δ mutation was introduced into a strain with a telomere reporter gene, no telomeric silencing defect was seen (data not shown). Thus, although Esc4 binds to Sir3, Esc4 does not appear to be a protein required for Sir protein-mediated silencing.
As shown in Figure 5, like esc4 Δ mutants, slx4 Δ mutants were sensitive to 0.032% MMS. Furthermore, the esc4 Δ slx4 Δ double mutant did not exhibit a greater MMS sensitivity than either single mutant (Figure 5A and data not shown), suggesting that they cooperate in providing resistance to MMS. Another group reported that an esc4 Δ slx4 Δ double mutant was more sensitive than either single mutant but the difference was very slight . In contrast, an esc4 Δ, but not a slx4 Δ mutant, was significantly HU-sensitive, and the double mutant was no more sensitive than the esc4 Δ strain. Thus, Esc4 appears to act independently of Slx4 in providing resistance to HU.
Because Esc4 bound to Slx4 and because the mutant was sensitive to MMS, this suggested that Esc4 might function in the same pathway as Slx4. SLX4 was first identified in a screen for genes required for viability of yeast cells deleted for SGS1 . Therefore, we tested if esc4 was also synthetically lethal with sgs1. To do this, an esc4 Δ mutant was crossed with an sgs1 Δ mutant, the diploid was sporulated and dissected and meiotic progeny were analyzed. Haploid esc4 Δ sgs1 Δ cells were viable, but were noticeably slower-growing that either single mutant (e.g., see Figure 5, YPD plate). While this work was in progress, this genetic interaction was also observed in genome-wide studies [41, 46].
An sgs1 Δ mutant showed sensitivity to both MMS and HU (Figure 5), as expected based on previously published results. An asf1 Δ mutant was used as a control and displayed sensitivity to both DNA damaging chemicals, as expected [57, 58]. Interestingly, an esc4 Δ sgs1 Δ mutant displayed MMS and HU sensitivity that was much more pronounced than that of either single mutant (Figure 5A and 5B). The enhanced sensitivity of this double mutant (10,000-fold on 0.014% MMS plates) seemed to be due to a synergistic repair defect and not entirely due to the growth defect (10-fold difference in colony number on YPD control plates) that was also observed in the esc4 Δ sgs1 Δ strain.
By screening a library of factors that could function in place of the HMR-E silencer when targeted to DNA, we identified Esc4 for its ability to e stablish s ilent c hromatin . Protein sequence analysis showed that Esc4 protein contains six BRCT motifs; four are found in tandem at the amino-terminus and two more are at the carboxy-terminus. The entire Esc4 protein was present in the hybrid identified in the targeted silencing screen. Since targeted silencing by Esc4 at HMR was found to be SIR-dependent, it seemed likely that some region within Esc4 was attracting a silencing protein complex to DNA. We tested subsets of the BRCT motifs, as well as the linker between them, for targeted silencing at HMR. These experiments demonstrated that the C-terminal two BRCTs caused targeted silencing that was nearly as strong as with full-length Esc4. Because silencing by this pair of BRCT motifs of Esc4 was also SIR-dependent, it seemed very likely that this region was recruiting a Sir protein when tethered to DNA. Therefore, we tested the C-terminal BRCT motifs for interactions with known silencing proteins by two-hybrid analysis. We identified a specific interaction with Sir3 (aa 252–978). We conclude that binding of Sir3 by Esc4 is likely to be responsible for the SIR-dependent targeted silencing activity.
In some cases BRCT motifs have been shown to bind to phosphorylated serine residues. Specifically, they have been shown to bind to phosphopeptides with the following consensus: pSxxF . Interestingly, Sir3 has an SxxF sequence (aa 583–586) within the Esc4-interacting region that we describe here (aa 252–978) and, furthermore, Sir3 protein has been shown to be phosphorylated [59, 60], suggesting that an Esc4 BRCT motif or perhaps the combination of the two in the C-terminus may bind to phospho-Sir3. However, not all proteins bound by BRCT motifs have the SxxF motif , so the precise BRCT-interacting region of Sir3 could be elsewhere.
In addition to binding Sir3 via C-terminal tandem BRCT motifs, Esc4 also binds to Slx4 via four tandem N-terminal BRCTs, as we have shown here by two-hybrid screening. This two-hybrid result demonstrates that these four BRCTs are sufficient for binding Slx4 and agrees with a recent report showing that the N-terminal BRCT motifs are required for this interaction . It seems quite possible that Esc4 could bind Sir3 and Slx4 concurrently, given that these nuclear proteins' binding sites within Esc4 map to BRCT clusters separated by a long linker. Slx4 has been shown to heterodimerize with the endonuclease Slx1 to cleave DNA containing 5' -flap structures, such as in stalled replication forks, to facilitate their repair . Thus, Esc4 binds the silencing protein Sir3 and also to Slx4, an important DNA repair complex component. Esc4 may play a role in facilitating repair of aberrant DNA structures, perhaps specifically within silent chromatin. Esc4 is a Mec1 kinase target  and this phosphorylation is required for its repair function. It is possible that phosphorylation of Esc4 by Mec1, which occurs just N-terminal to the Sir3-binding BRCTs, regulates association with Sir3 or other factors required for its ability to repair particular chromosomal loci in S phase.
We analyzed Esc4 protein alignments (such as that shown in Figure 3) for evidence of conserved regions in the protein other than BRCT motifs. One region of interest was the SQ/TQ motifs between amino acids 743 and 807, which were shown to be important for function in DNA repair . We did not find that these motifs were well-conserved, suggesting that the specific site of phosphorylation is not particularly critical in proteins with otherwise similar overall BRCT domain architecture (i.e., [BRCT]4-linker-[BRCT]2 arrangement). This may be because of some differences in Esc4 functions in diverse yeasts or may suggest that flexibility is tolerated in positioning of the phosphorylation sites, and therefore the exact relative location of kinase target sites has not been constrained during evolution.
Future structural and genome sequencing studies are likely to unveil similarities and differences between multi-BRCT domain containing proteins. Whether these proteins play largely protein-scaffolding roles or also contain intrinsic enzymatic properties will be interesting to discover.
We have shown that Esc4 caused targeted silencing when tethered at a weakened HMR locus. The targeted silencing activity was primarily due to the C-terminal two tandem BRCT motifs in Esc4, which bound to Sir3, probably through a direct interaction. This interaction led to the recruitment of the Sir complex and hence caused targeted silencing. The N-terminal BRCT domains were sufficient for binding to Slx4, which functions with Esc4 in DNA repair. Thus, the nuclear Esc4 protein uses its six BRCT motifs to connect diverse proteins involved in DNA repair and silent chromatin.
Esc4 was identified in a targeted silencing screen that has been described previously . Briefly, a Gal4 DNA binding domain (GBD) library was screened for hybrid proteins capable of establishing silencing of a URA3 reporter gene integrated in place of mating-type genes at an HMR locus that had the HMR-E silencer replaced by a Gal4 DNA binding site (G) (see Figure 1A). A full length GBD-Esc4 (1–1070) clone, aeb15, was identified as being capable of establishing targeted silencing of hmr::URA3, causing resistance to 5-fluororotic acid (5-FOA). This GBD-Esc4 clone was subsequently transformed into strain YEA76 (HMR-E Aeb::G hmr::URA3 gal4::LEU2) and YEA77 (HMR-E aeB::G hmr::URA3 gal4::LEU2) (see Table 1 and Yeast Strain section for details) and tested for targeted silencing. To test SIR-dependence of targeted silencing by Esc4 at HMR, shown in Figure 4, targeted silencing in strain YEA76 (SIR+) was compared with that in sir mutant derivatives YAM7 (sir2 Δ), YEA 118 (sir3 Δ), and YKAB17 (sir4 Δ).
For the targeted silencing experiments shown in Figures 2 and 4, assays were carried out as follows: strains were transformed with plasmids expressing the appropriate GBD hybrid protein, grown at 30°C for two days in SC -Trp medium (to select for the GBD plasmid), serially diluted ten-fold five times and spotted on SC -Trp + 5-FOA plates (to assay for silencing at HMR) or on SC -Trp control plates.
Plasmid aeb15, expressing GBD-Esc4(1–1070) was isolated in the targeted silencing screen. This plasmid was recovered from a library based on pGBT9.C (ADH1 UAS -G BD , TRP1, CEN4/ARS1; see Acknowledgements). To generate plasmids for use in the two-hybrid system, ESC4 sequences were amplified from genomic DNA and subcloned into plasmid pSTT91, a derivative of pBTM116 that contains the ADE2 gene (; see also ). Plasmids pAM2 (LexA-Esc4N (a.a. 1–480) and pAM7 (LexA-Esc4C (a.a. 818–1070) were used for two-hybrid experiments. To test for LexA-Esc4C binding to GAD-Sir3 and GAD-Sir4, plasmid pEDA195, GAD-Sir3(252–978), and pCTC36, GAD-Sir4(839–1358), were used. pCTC36 expresses the same region of SIR4 as plasmid pCTC18  except that the Sir4 hybrid is expressed from pGAD-R. pEDA195 was constructed by cloning a Pst I fragment of the SIR3 gene into vector pGAD424. Plasmid pAM2, expressing LexA-Esc4(1–480), and a clone expressing GAD-Slx4(383–748) isolated in the two-hybrid screen were used as a positive control in two-hybrid experiments summarized in Table 3.
Yeast strain construction
All strains are listed in Table 1. To make slx1 Δ, slx4 Δ, sir2 Δ, sir4 Δ, and sgs1 Δ mutants, PCR primers with 5' homology to sequences flanking these ORFs and 3' homology to sequences in a plasmids harboring selectable marker genes were used for PCR, generating targeting cassettes that were transformed into yeast as has been previously described . esc4 Δ ::his5+ mutants were generated by the same method, but using a different plasmid as a template for PCR (gift of N. Dean, Stony Brook University). Strain YEA76 and its derivatives are derived from strain YSB35 .
Two-hybrid screening and direct tests
Screening was performed essentially as described [54, 63]. Plasmid pAM2, which expresses LexA-Esc4(1–480), (ADE2, TRP1, 2 μ) was co-transformed with approximately 1 μg of GAD library (LEU2, 2 μ; ) into strain L40 , which contains LexA binding sites upstream of both the HIS3 gene and the LacZ gene. The following specificity tests were performed: (1) His+ candidates were assayed for lacZ expression both after curing candidates of either the LexA or the GAD plasmid, (2) GAD hybrids were tested for interactions with nonspecific LexA hybrids (e.g. LexA-lamin) by mating candidates cured of the bait plasmid to LexA-containing AMR70 , and (3) the GAD hybrid plasmids which passed the aforementioned tests were subsequently retransformed into L40 along the original bait and tested for LacZ expression.
Two-hybrid tests done using the C-terminal BRCT motifs in Esc4 were performed by co-transforming L40 with plasmid pAM7, expressing LexA-Esc4(818–1070), and various GAD hybrids to silencing proteins, such as Sir1, Sir2, Sir3, Sir4, and Rap1. To test whether Sir3 bridged binding of LexA-Esc4(818–1070) to Sir4, a sir3Δ::kanMX6 derivative of L40 was generated (see yeast strains section for details), strain YRJ3, and the interaction was then retested in this strain. The control sir4 Δ-derivative of L40 used was strain YJL03.
DNA damage sensitivity tests
Cultures were grown in YPD medium ~18 h at 30°C and then serially diluted ten-fold, five times before being spotted onto plates containing YPD medium with MMS, HU, or no chemical. Cells were then incubated at 30°C and, in the case of MMS media, the plates were wrapped in aluminum foil.
We thank Carlos Evangelista and Stan Fields for the G BD library and Phil James for the two-hybrid GAD library. Thanks to Joe Landry for strain YJL103, Erik Andrulis for plasmid pEDA195 and to Cheng-ting Chien for pCTC36. Aaron Neiman and Doug Theobald provided helpful suggestions. This work was supported by NIH grant GM28220 and a Carol Baldwin Breast Cancer Research Support Grant.
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