Histone H2A‐H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin
2018; Springer Nature; Volume: 37; Issue: 19 Linguagem: Inglês
10.15252/embj.201899021
ISSN1460-2075
AutoresCécile Evrin, Joseph D. Maman, Aurora Diamante, Luca Pellegrini, Karim Labib,
Tópico(s)Plant Molecular Biology Research
ResumoArticle13 August 2018Open Access Transparent process Histone H2A-H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin Cecile Evrin Cecile Evrin MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Joseph D Maman Joseph D Maman Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Aurora Diamante Aurora Diamante Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Luca Pellegrini Luca Pellegrini orcid.org/0000-0002-9300-497X Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Karim Labib Corresponding Author Karim Labib [email protected] orcid.org/0000-0001-8861-379X MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Cecile Evrin Cecile Evrin MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Joseph D Maman Joseph D Maman Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Aurora Diamante Aurora Diamante Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Luca Pellegrini Luca Pellegrini orcid.org/0000-0002-9300-497X Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Karim Labib Corresponding Author Karim Labib [email protected] orcid.org/0000-0001-8861-379X MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Author Information Cecile Evrin1, Joseph D Maman2, Aurora Diamante2, Luca Pellegrini2 and Karim Labib *,1 1MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK 2Department of Biochemistry, University of Cambridge, Cambridge, UK *Corresponding author. Tel: +44 1382 384108; E-mail: [email protected] The EMBO Journal (2018)37:e99021https://doi.org/10.15252/embj.201899021 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The eukaryotic replisome disassembles parental chromatin at DNA replication forks, but then plays a poorly understood role in the re-deposition of the displaced histone complexes onto nascent DNA. Here, we show that yeast DNA polymerase α contains a histone-binding motif that is conserved in human Pol α and is specific for histones H2A and H2B. Mutation of this motif in budding yeast cells does not affect DNA synthesis, but instead abrogates gene silencing at telomeres and mating-type loci. Similar phenotypes are produced not only by mutations that displace Pol α from the replisome, but also by mutation of the previously identified histone-binding motif in the CMG helicase subunit Mcm2, the human orthologue of which was shown to bind to histones H3 and H4. We show that chromatin-derived histone complexes can be bound simultaneously by Mcm2, Pol α and the histone chaperone FACT that is also a replisome component. These findings indicate that replisome assembly unites multiple histone-binding activities, which jointly process parental histones to help preserve silent chromatin during the process of chromosome duplication. Synopsis Replisome assemblies unite multiple histone-binding activities that jointly process parental histones during DNA replication. Among them, polymerase α has a particular role via histone H2A-H2B binding to preserve gene silencing at budding yeast telomeres and mating-type loci. Ctf4-dependent Pol α replisome tethering is dispensable for efficient DNA synthesis but required for maintenance of repressive chromatin. The Pol α catalytic subunit contains a conserved histone-binding motif similar to the motif in Mcm2 helicase subunit. Unlike H3-H4 binding by Mcm2, the Pol α motif binds histones H2A-H2B. Mutation of either Pol α or Mcm2 histone-binding motifs abrogates gene silencing at telomeres and mating-type loci. Introduction A subset of the many factors required to duplicate chromosomes assembles into a large and highly dynamic molecular machine called the replisome (Bell & Labib, 2016; Burgers & Kunkel, 2017; Kunkel & Burgers, 2017; Riera et al, 2017). Bacterial, viral and eukaryotic replisomes vary widely in their composition, but a common feature in all cases is that replisome assembly connects the DNA helicase that unwinds the parental DNA duplex to one or more of the DNA polymerases that synthesize the two daughter strands. Such physical connections between helicase and polymerases can be direct or else be mediated by other factors, and can serve to couple the rates of DNA unwinding and DNA synthesis, thus minimizing the exposure of single-strand DNA at replication forks. In addition, work with Escherichia coli showed that replisome assembly allows DNA polymerase action to propel the DNA helicase forwards and thus stimulate the rate of fork progression (Kim et al, 1996). Whereas the E. coli replisome is well characterized and the role of each component is defined, the eukaryotic replisome remains an enigma and contains around twice as many subunits, many of which are still of unknown biochemical function. The greater complexity of the eukaryotic replisome reflects the additional challenges of duplicating chromosomes that contain vast amounts of DNA packaged with histones into chromatin, which must be disrupted and then re-established during replication, whilst preserving epigenetic information that controls patterns of gene expression (Hammond et al, 2017; Miller & Costa, 2017). Very little is known about the activities within the replisome that contributes to the processing of parental chromatin during chromosome duplication. Moreover, the links between parental histone processing at replication forks and the preservation of gene silencing are largely unexplored. The eukaryotic replisome connects the CMG helicase (Cdc45-MCM-GINS) to two of the three DNA polymerases that are essential for DNA synthesis at eukaryotic replication forks, namely DNA polymerase epsilon (Pol ε) that synthesizes the leading strand and DNA polymerase alpha (Pol α) that initiates each new Okazaki fragment during lagging strand synthesis. Pol ε binds directly to the CMG helicase and stimulates the rate of fork progression (Kang et al, 2012; Sengupta et al, 2013; Georgescu et al, 2014; Langston et al, 2014), analogous to the stimulation of helicase by polymerase in the E. coli replisome (Kim et al, 1996). In contrast, Pol α is connected indirectly to the CMG helicase by the trimeric adaptor known as Ctf4 (Zhu et al, 2007; Gambus et al, 2009; Tanaka et al, 2009a), each protomer of which can bind to short "Ctf4-Interacting Peptides" (or CIP-boxes) in client proteins such as the Sld5 subunit of yeast CMG, or the Pol1 catalytic subunit of Pol α (Simon et al, 2014; Villa et al, 2016). The tethering of Pol α to the eukaryotic replisome by Ctf4 was originally thought to promote efficient priming of Okazaki fragments during lagging strand DNA synthesis. However, Ctf4 has no apparent impact on DNA synthesis in vitro, using a reconstituted replisome system based on purified budding yeast proteins (Yeeles et al, 2015, 2017). Moreover, recent work indicates that trimeric Ctf4 represents a hub in the eukaryotic replisome with roles that go beyond DNA synthesis (Fumasoni et al, 2015; Samora et al, 2016; Villa et al, 2016). In addition to Pol α, budding yeast Ctf4 recruits other CIP-box proteins such as the Chl1 helicase that helps to establish sister chromatid cohesion (Samora et al, 2016) and the Dna2 and Tof2 proteins that help to maintain the integrity of the rDNA repeats on chromosome 12 (Villa et al, 2016). Here, we show that replisome tethering of Pol α via Ctf4 is dispensable for efficient DNA synthesis in budding yeast cells, and instead is required to preserve epigenetic gene silencing at telomeres and the silent mating-type genes, in a manner that is dependent upon a novel histone-binding motif in the amino-terminal region of the Pol1 DNA polymerase subunit. These findings expand our view of the eukaryotic replication machinery and show how the coupling of a DNA polymerase to the helicase within the replisome can contribute to functions beyond DNA synthesis. Results Displacement of Pol α from Ctf4 does not perturb DNA replication in vivo We previously showed that Pol α was no longer able to associate with Ctf4 in pol1-4A cells with mutations in the CIP-box motif of the catalytic subunit (Simon et al, 2014). However, pol1-4A cells lack multiple phenotypes of ctf4∆ strains, which instead reflect the recruitment by Ctf4 of other CIP-box proteins to the replisome (Samora et al, 2016; Villa et al, 2016). To examine in more detail the consequences of displacing Ctf4-tethered Pol α from the replisome, we synchronized control cells and pol1-4A in G1-phase and monitored DNA synthesis and replisome assembly when cells entered S-phase. As shown in Fig 1, the kinetics of DNA synthesis were very similar in pol1-4A and control cells (Fig 1A), and replisome assembly was normal except that Pol α was largely displaced (Fig 1B; Appendix Fig S1 shows that the weakened association of Pol1-4A with the replisome is equivalent to the situation previously reported for ctf4∆ cells (Sengupta et al, 2013), reflecting a residual Ctf4-independent link between Pol α and the CMG helicase). Figure 1. Ctf4-dependent tethering of Pol alpha to the replisome is dispensable for efficacious DNA synthesis in budding yeast cells Control (YCE542) and pol1-4A (YCE544) cells were synchronized in G1-phase at 24°C then released into S-phase for the indicated times. DNA content was measured by flow cytometry throughout the experiment. In a similar experiment to that described above, samples from the G1-phase and 30′ S-phase timepoints were used to prepare cell extracts, from which the CMG helicase component Sld5 was isolated by immunoprecipitation. The associated replisome components were monitored by immunoblotting. The asterisk indicates a non-specific band in the anti-Pob3 immunoblot (corresponding to TAP-Sld5). Diploid cells of the indicated genotype were sporulated, and then, asci were dissected on rich medium. The image of the resultant tetrads was taken after 2 days growth at 30°C, and the genotype of each colony was determined by replica plating to selective media, indicating that the growth of pol1-4A is not affected by combination with mec1∆. Equivalent analysis showing that pol1-F1463A is synthetic lethal with mec1∆. Download figure Download PowerPoint As a more sensitive assay for impaired DNA synthesis, we combined pol1-4A with deletion of the MEC1 gene, the yeast orthologue of the ATR checkpoint kinase, which becomes essential in cells that have the slightest defect in DNA replication. Colony growth of mec1∆ cells was unaffected by the pol1-4A mutations (Fig 1C), indicating that Pol α is still very efficient at priming nascent DNA during leading and lagging strand synthesis in pol1-4A cells, even though it is no longer tethered to the replisome by Ctf4. As a control, we confirmed that mec1∆ is synthetic lethal with the pol1-F1463A point mutation (Fig 1D), which displaces primase from the carboxyl terminus of Pol1 (Kilkenny et al, 2012). Previous work indicated that the progression of chromosome replication in pol1-F1463A is very similar to control cells, but cell viability is absolutely dependent upon Mec1, pointing to a subtle defect in DNA synthesis at replication forks (Kilkenny et al, 2012). Overall, these findings indicated that replisome tethering of Pol α by Ctf4 is dispensable in vivo for efficient DNA synthesis and instead fulfils a different role during chromosome replication. Replisome tethering of Pol α is required to preserve gene silencing at sub-telomeric and mating-type loci One possible function for Ctf4-dependent replisome tethering of Pol α was suggested by analogy with the rDNA-associated protein Tof2 and the Dna2 nuclease, which are recruited to the replisome by Ctf4 as part of a mechanism that preserves the integrity of the large array of rDNA repeats (Villa et al, 2016). As seen for Pol α, mutation of the CIP-boxes of Tof2 and Dna2 disrupted their interaction with Ctf4 but did not perturb DNA replication in yeast cells. Instead, the tof2-4A and dna2-4A alleles both led to a reduction in the size of chromosome 12 (Villa et al, 2016). However, this phenotype was not observed with pol1-4A cells (CE and KL, unpublished data), indicating that Ctf4 tethers Pol α to the replisome in order to support some other function. A second possibility was indicated by the observation that Pol α co-purifies from yeast cell extracts with histone complexes that have been released from chromatin (Foltman et al, 2013). This suggested that Pol α might have an associated histone-binding activity that contributes to chromatin replication when Pol α is tethered to the replisome by Ctf4, analogous to the role of the histone-binding motif of the Mcm2 subunit of the CMG helicase (Ishimi et al, 1998; Foltman et al, 2013; Huang et al, 2015; Richet et al, 2015), which is required to preserve gene silencing near budding yeast telomeres (Foltman et al, 2013). Similar to the phenotype of pol1-4A cells, mutation of the conserved histone-binding motif of Mcm2 is not synthetic lethal with mec1∆ (Foltman et al, 2013) and thus does not perturb DNA replication in yeast cells. If the ADE2 marker gene is placed near a telomere in a wild-type yeast strain, it is expressed in some cells and repressed in others for many generations (Fig 2A), leading to colonies with white sectors (expressing ADE2) and red sectors (repression of ADE2). However, when a haploid strain with telomeric ADE2 was crossed to the mcm2-3A histone-binding mutant and the resulting diploid strain was sporulated, the mcm2-3A progeny that inherited the marker gene produced pure white colonies (Foltman et al, 2013), in which all cells express sub-telomeric ADE2 (Fig 2B). Strikingly, we found in similar experiments that pol1-4A is also defective in the maintenance of telomeric silencing (Fig 2C), and the same is true for ctf4∆ cells (Suter et al, 2004). Moreover, this phenotype was not produced by mutation of the CIP-box in other Ctf4 partners such as Tof2 or Dna2 (Fig EV1). These findings indicated that Ctf4-dependent tethering of Pol α to the replisome is required to preserve gene silencing near a telomere, but is not required for Pol α to fulfil its role in DNA synthesis. Consistent with this view, telomere length is normal in pol1-4A cells (Appendix Fig S2; compare pol1-4A and control), whereas telomere length increases as a consequence of defects in Pol α catalytic function (Adams & Holm, 1996), or following the displacement of primase from Pol α (Appendix Fig S2, pol1-F1463A). Figure 2. Displacement of Pol alpha from the replisome leads to a loss of gene silencing at sub-telomeric or mating-type loci A–C. Tetrad analysis of meiotic progeny of the indicated diploids (heterozygous for sub-telomeric ADE2), processed as described in Materials and Methods. Sectored colonies represent epigenetic variation in the expression of the ADE2 marker at the sub-telomeric location. White colonies indicate expression of ADE2 in all cells, and red colonies lack the ADE2 marker (as denoted in the right-hand panels). We examined a total of 19 mcm2-3A VR::ADE2-TEL colonies and 11 pol1-4A VR::ADE2-TEL colonies. D–F. Equivalent analysis of the meiotic progeny of diploid cells that were heterozygous for an insertion of the ADE2 marker at HMR locus on chromosome 3 (denoted HMR::2EDA since the orientation of the ADE2 marker was such that the promoter was distal to the HMR-E silencer element). We examined a total of 19 mcm2-3A HMR::2EDA colonies and 12 pol1-4A HMR::2EDA colonies. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Sub-telomeric silencing is not affected by CIP-box mutations in the Ctf4 partners Dna2 or Tof2, or by deletion of the TEL1 geneThe indicated diploids were processed as described above for Fig 2. Download figure Download PowerPoint Gene silencing in budding yeast cells also occurs at the silent mating-type genes on chromosome 3 (Haber, 2012). The ADE2 marker gene is very strongly repressed when inserted at the HMR mating-type locus in control cells and therefore produces red colonies that are only slightly paler than colonies of control cells (Fig 2D; ADE2 replaces the silenced MATa2 gene and has the same orientation, designated HMR::2EDA). However, we found that HMR-2EDA is largely de-repressed in mcm2-3A and pol1-4A cells, which thus grew as very pale pink colonies (Fig 2E and F; note that the control HMR-2EDA cells sometimes produced dark pink rather than red colonies in this experiment, probably due to partial de-repression of HMR-2EDA in the parental diploid cells that were heterozygous for mcm2-3A or pol1-4A). These data indicate that the histone-binding activities of the replisome are important to preserve gene silencing at the mating-type loci, in addition to being important for telomeric silencing (Fig EV2B shows that the same is true in HMR::ADE2 cells in which ADE2 is inserted in the opposite orientation to HMR::2EDA, leading to weaker gene silencing). Click here to expand this figure. Figure EV2. Replisome-tethered histone-binding activity of Mcm2 and Pol alpha is required for gene silencing at the HMR mating-type locus A–E. Tetrad analysis of meiotic progeny of the indicated diploids (heterozygous for insertion of the ADE2 marker gene at the HMR locus on chromosome 3), processed as described in Materials and Methods. The promoter of the ADE2 marker was positioned proximal to the HMR-E silencer element (denoted HMR::ADE2), and this corresponded to weaker silencing of ADE2 compared to the HMR::2EDA strains in Fig 2D–F. We examined a total of 16 mcm2-3A HMR::ADE2 colonies, 16 pol1-4A HMR::ADE2 colonies, 15 pol1-2A2 HMR::ADE2 colonies and 16 pol1-6A HMR::ADE2 colonies. Download figure Download PowerPoint Finally, we tested whether the mcm2-3A and pol1-4A mutations affected silencing of a URA3 marker gene that had been inserted into one copy of the rDNA repeats on chromosome 12. Interestingly, gene silencing in the rDNA is mechanistically distinct from silencing at telomeres and the silent mating-type loci (Srivastava et al, 2016). The URA3 gene was silenced when inserted into either the NTS1 or NTS2 sequences of an rDNA repeat, and silencing at NTS1 was relieved in cells that lack the type I topoisomerase Top1 (Fig EV3), as reported previously (Huang et al, 2006). However, gene silencing at NTS1 and NTS2 persisted in mcm2-3A and pol1-4A cells. Therefore, although Mcm2 and replisome-tethered Pol α are both required to preserve gene silencing at telomeric and mating-type loci in budding yeast cells, they are dispensable for the mechanistically distinct phenomenon of rDNA silencing. Click here to expand this figure. Figure EV3. Histone-binding activities of replisome proteins are largely dispensable for gene silencing within the rDNA repeatsSerial dilutions of the indicated strains, in which the "modified URA3 marker" (mURA3) was inserted at the leu2 locus or else within the "Non-transcribed spacers" of an rDNA repeat (NTS1 and NTS2), were grown on non-selective or selective medium, as described in Materials and Methods. In control cells, mURA3 can be expressed from the leu2 locus but is repressed when present at the NTS1 or NTS2 element of an rDNA repeat. Repression at NTS1 was lost in top1∆ cells, as seen previously (Huang et al, 2006), but an equivalent defect was not observed in mcm2-3A, pol1-2A2, pol1-4A, pol1-6A, mcm2-3A pol1-6A, dpb3∆ or dpb4∆. Download figure Download PowerPoint The amino terminus of the DNA polymerase subunit of Pol α contains a conserved histone-binding motif The ability of Pol α to interact in yeast cell extracts with chromatin-derived histone complexes might reflect a novel histone-binding activity in Pol α, but could also be due to the previously described association of Pol α with the histone chaperone FACT (Miles & Formosa, 1992). Consistent with the former possibility, we noticed that the extended amino-terminal tail of the Pol α catalytic subunit in eukaryotes contains a motif that resembles the histone-binding element in Mcm2 (Fig 3A), in that it contains two conserved aromatic residues that are separated by eight amino acids and are flanked by conserved acidic residues, adjacent to a predicted alpha helix (Foltman et al, 2013). However, the two motifs also have important differences, since the orientation of the alpha helix and the adjacent pair of aromatic residues is reversed between Mcm2 and Pol α, and the Pol1 tail has an additional pair of conserved tyrosines that overlap with the predicted alpha helix. Figure 3. The amino terminus of Pol1 binds chromatin-derived histone complexes, together with Mcm2 and FACT Sequence alignment indicating the location of the CIP-box and a newly identified putative histone-binding motif in the amino-terminal tail of budding yeast Pol1. Asterisks under the CIP-box indicate residues mutated in the pol1-4A allele, whereas those under the novel histone-binding motif denote two conserved aromatic residues separated by eight amino acids, as in the histone-binding motif of Mcm2. Control cells (YSS3), cells expressing ProteinA-tagged Pol1NT (YCE39) and cells expressing ProteinA-tagged Pol1NT-4A (YCE217) were synchronized in G2-M phase at 30°C, before the isolation of the ProteinA-tagged Pol1 tails on IgG beads. Pol1NT interacts with Ctf4, dependent upon the CIP-box, but does not interact with FACT (for which the Spt16 subunit is shown). Cells expressing ProteinA-tagged Pol1NT (YCE39) were grown as above, but the resultant extracts were then incubated +/−DNase, before isolation of Pol1NT. The associated proteins were monitored by immunoblotting—the asterisk in the Pob3 blot indicates faint cross-reactivity with ProteinA-Pol1NT. Analogous experiment comparing expression of GAL-Pol1NT in control cells (YCE39) or mcm2-3A cells (YCE238). After release of histones from chromatin by DNase treatment, the co-purification of Mcm2 with Pol1NT depends on the H3-H4 binding motif of Mcm2, which is mutated in mcm2-3A. Similar experiment comparing expression of GAL-Pol1NT in cells with a second copy of SPT16 at the leu2 locus, expressing either wild-type Spt16 (YCE248) or Spt16 with a small deletion at the carboxyl terminus (YCE250) that abolishes interaction of FACT with H3-H4 tetramers. Cultures of GAL-Pol1NT (YCE39), MCM2-9MYC (YMP154-1) and SPT16-9MYC (YMP177-1) were arrested in G2-M phase at 30°C, and then, equal volumes were mixed as indicated. Cell extracts were prepared, and Pol1NT was isolated on IgG-coated magnetic beads. Release of histones from chromatin by DNase treatment allowed Pol1NT from the first culture to form ternary complexes containing not only histones but also Mcm2-9MYC or Spt16-9MYC from the second cultures. Download figure Download PowerPoint To test the ability of the amino-terminal tail of budding yeast Pol1 to associate with histone complexes and FACT, we expressed the first 351 amino acids of Pol1 in yeast cells as a fusion to Protein A, and then isolated the fusion protein from cell extracts, with or without prior DNase treatment to release histone complexes from chromatin. As shown in Fig 3B, the amino-terminal fragment of Pol1 (hereafter termed Pol1NT) associated specifically with Ctf4, dependent upon the integrity of the CIP-box (Fig 3B, compare Pol1NT-4A with wt Pol1NT), but did not associate with FACT, which must instead bind to some other region of Pol α. However, upon digestion of genomic DNA to release histone complexes from chromatin, Pol1NT co-purified not only with histones but also with FACT in yeast cell extracts (Fig 3C, +DNase; Appendix Fig S3 shows the salt sensitivity of the observed complexes). These findings suggested that Pol1NT shares the ability of Mcm2 to associate with histone complexes that have been released from chromatin and also indicated that the same histone-containing complexes can be bound by FACT. To explore this further, we expressed Pol1NT (Pol1 1–351) and Mcm2NT (Mcm2 1–200) in yeast cells as fusions to Protein A and also included Protein A-fused Mcm4NT (Mcm4 1–186) as a negative control. We isolated the fusion proteins by immunoprecipitation from yeast extracts with or without DNase treatment, before analysis of the associated factors by mass spectrometry. As summarized in Table 1, the treatment of yeast extracts with DNase greatly stimulated the detectable association of both Pol1NT and Mcm2NT, but not Mcm4NT, with histones and FACT. In addition, Mcm2 (but not Mcm4) was greatly enriched in the Pol1NT immunoprecipitates after DNase treatment of extracts. Incidentally, we found that the Tra1 subunit of the SAGA and NuA4 histone acetyltransferases was also enriched in the immunoprecipitates of Pol1NT and Mcm2NT after DNase treatment, suggesting that Tra1 might also bind to histone complexes released from chromatin, presumably on distinct sites to Mcm2NT and Pol1NT. Table 1. Mass spectrometry analysis of proteins co-purifying with Pol1NT, Mcm2NT or Mcm4NT, +/−DNase treatment to release histone complexes from chromatin Identified protein MS analysis of IPs from G2-M cell extracts (spectral counts) Pol1 (1–351) +DNase Pol1 (1–351) −DNase Mcm2 (1–200) +DNase Mcm2 (1–200) −DNase Mcm4 (1–186) +DNase Pol1NT 1,570 854 7 0 0 Mcm2 436 0 258 218 6 Mcm4 38 11 14 14 681 Ctf4 938 564 0 0 0 Tel1 407 229 18 0 0 Histone H2A 95 0 46 0 16 Histone H2B 441 3 140 3 33 Histone H3 275 2 178 5 13 Histone H4 287 4 275 4 28 Spt16 2,255 53 1,558 5 201 Pob3 812 8 486 0 46 Tra1 600 32 1,105 44 86 Spt5 396 22 38 3 26 The indicated protein fragments were expressed in G2-M-arrested cells and then isolated by immunoprecipitation on IgG-coated magnetic beads. The resultant material was resolved in a 4–12% gradient gel, which was then stained with colloidal Coomassie blue, before each lane was cut into 40 bands for mass spectrometry analysis. The table summarizes the spectral counts that were detected for each factor that specifically co-purified with the indicated fragment. As expected, we found that Pol1NT interacted specifically with Ctf4 regardless of DNase treatment. Moreover, Pol1NT interacted with the Tel1 checkpoint kinase in a similar manner to Ctf4 (Table 1), indicating that Tel1 is a novel partner of Pol1NT. However, subsequent experiments indicated that neither Ctf4 (Fig EV4A) nor Tel1 (Fig EV4B) was required for Pol1NT to associate with histone complexes released from chromatin. Consistent with this, tel1∆ was not associated with a loss of telomeric silencing (Fig EV1), though it did reduce telomere length (Appendix Fig S2), as previously reported (Lustig & Petes, 1986; Greenwell et al, 1995). Click here to expand this figure. Figure EV4. Neither Ctf4 nor Tel1 is required for the histone-binding activity of Pol1NT Mutation of the CIP-box of Pol1 displaces Ctf4 without affecting the interaction with histones released from chromatin. Although Tel1 is a novel partner of Pol1NT, deletion of the TEL1 gene does not affect histone-binding by Pol1NT. Download figure Download PowerPoint The presence of Mcm2 in the Pol1NT immunoprecipitates was dependent not only upon the release of histones from chromatin by DNase treatment, but also required the integrity of the conserved histone-binding motif in the Mcm2 tail (Fig 3D, mcm2-3A). Similarly, the co-purification of FACT with Pol1NT, upon release of histones from chromatin, was abolished by mutation of the carboxyl terminus of the Spt16 subunit (Fig 3E and Appendix Fig S4), corresponding to a small truncation that was previously shown to abolish the association of FACT with H3-H4 tetramers (Tsunaka et al, 2016). These findings indicated that Pol1NT, Mcm2NT and FACT are able to bind to different regions of the same histone complexes that are released from chromatin. To confirm that these interactions can indeed occur in vitro upon release of histone complexes from DNA, we mixed a yeast culture expressing Pol1NT with cells expressing either Mcm2-9MYC or Spt16-9MYC, before making mixed cell extracts. In the absence of DNase treatment, Pol1NT from the first cell culture did not interact with either Mcm2-9MYC or SPT16-9MYC from the second cultures (Fig 3F, −DNase). In contrast, DNase treatment of the cell extracts allowed Pol1NT from the first culture not only to associate in vitro with histones, but also to co-purify with tagged Mcm2 or Spt16 from the second culture (Fig 3F, +DNase). Overall, these findings indicated that Pol1NT is able to interact in vitro with chromatin-derived histone complexes, which can also be co-chaperoned by Mcm2 and FACT. Mutation to alanine of six conserved residues in Pol1NT (Y37, Y45, F58, D62, G66 and Y67), within the region of similarity with the histone-binding
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