Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1
Highlights
► Quantitative chromatin immunoprecipitation experiments confirm that Rad4TopBP1 associates with origins of DNA replication and, in addition, demonstrate that the protein is not present within the active replisome. ► A direct interaction between Rad4TopBP1 and Mcm10 is shown and this is reflected in the Rad4TopBP1-dependent origin association of Mcm10. ► Rad4TopBP1 chromatin association at stalled replication forks was found to be dependent upon the checkpoint protein Rad9 but importantly was independent of the Rad17 checkpoint protein.
Introduction
In order to achieve the faithful and complete replication of the genome eukaryotic cells initiate a single round of DNA replication per cell cycle at multiple sites, DNA replication origins, within the genome. DNA replication per se requires the activity of a number of DNA polymerases, the initial activity of which is confined to these origins [1]. The co-ordination of DNA replication with cell cycle progression requires exquisite regulation of the initiation of DNA replication by the cell cycle control machinery. This control is partly manifest as a stepwise, ordered assembly of a number of different factors at each individual origin of replication [2]. The hexameric origin recognition complex (ORC), consisting of the related Orc1-6 subunits is assembled first. Cdc6 and Cdt1 can then mediate recruitment of the MCM (mini-chromosome maintenance) heterohexamer Mcm2–7 to the replication origin through interaction with ORC. Formation of this pre-replication complex (pre-RC) prior to S phase licenses origins for replication, but initiation of DNA replication requires the activity of two protein kinases, CDK (cyclin dependent kinase) and DDK (Dbf4 dependent kinase), and the assembly of other factors including Cdc45, GINS, Mcm10, Sld2 and Sld3. In budding yeast Cdc45 and Sld3 associate with early firing origins in G1, prior to CDK and DDK activity, and with late firing origins in S phase [3], [4]. Evidence from budding yeast has demonstrated that Sld2 and Sld3 recruitment are pivotal events in replication initiation at origins and the two proteins are critical targets of the S phase CDK [5], [6], [7], [8]. Upon phosphorylation by CDK, Sld2 and Sld3 interact with distinct BRCT domains of a third protein, Dpb11, the Saccharomyces cerevisiae orthologue of human TopBP1, and that interaction enables recruitment to the pre-RC [6], [7], [8]. Phosphorylation of Sld2 promotes formation of a pre-loading complex containing Dpb11, the heteromeric GINS complex, DNA Polɛ and Sld2 itself [9]. The metazoan orthologue of Sld3, Treslin/Ticrr, has been shown to interact with TopBP1 and to be required for DNA replication [10], [11], [12]. Cdc45 recruitment appears to require direct interaction with Mcm complex and the mcm5-bob1-1 mutation results in DDK-independent loading of Cdc45 and the bypass of the DDK requirement for initiation of DNA replication [13], [14], [15]. Cdc45 loading exhibits interdependence with Sld3, which in the light of the data discussed above, offers a compelling mechanism for combining the CDK and DDK activities in the initiation of DNA replication [3]. Sld3 interacts with and is required for loading of the GINS complex in budding yeast [16]. In turn the GINS complex has been reported to be necessary for Cdc45 S phase chromatin association suggestive of an intimate, conserved functional relationship between Sld3, Cdc45 and GINS [16], [17]. More recent evidence demonstrates that GINS is dispensable for Cdc45 origin loading but is required for its interaction with the active replisome whilst Sld3 is required for Cdc45 and GINS origin loading but does not move with the replisome [4]. Mcm10 is also required for the initiation of DNA replication and is recruited to origins in a Mcm4-dependent manner, travels with the progressing replication fork and acts in part to maintain DNA polymerase α stability [18], [19], [20].
The picture of pre-replication complex assembly in the fission yeast, Schizosaccharomyces pombe is similar to that described above. One apparent difference is that fission yeast Sld3 loading is independent of Cdc45 rather than the loading of the two factors being interdependent [21]. Chromatin association of Cdc45 has been shown to be dependent upon Mcm10, DDK activity and Rad4/Cut5TopBP1 (hereafter Rad4TopBP1) function [22], [23]. DNA replication origin association of Rad4TopBP1 and Cdc45 were found to be GINS dependent in contrast to Sld3 loading [24]. Apart from these observations there has been comparatively little investigation into the role of Rad4TopBP1 at DNA replication origins in fission yeast. A number of studies have demonstrated that Rad4TopBP1 has dual functionality in the initiation of DNA replication and DNA damage checkpoint responses [25], [26], [27], [28], [29]. In addition to the observations described above the protein has been shown to interact with DNA polymerases δ and α [29]. Separation of DNA replication and checkpoint function mutations have been described and Rad4TopBP1 shown to be required for the activation of checkpoint kinases, Cds1 and Chk1 [28], [29], [30], [31] Rad4TopBP1 interacts with phosphorylated Rad9p, part of the 9-1-1 checkpoint clamp complex. This association is required for checkpoint-dependent Chk1 activation, as well as a number of other checkpoint proteins [29], [31]. Human TopBP1 has also been shown to function in both DNA replication and the response to DNA damage and recent data mirror the requirement for interaction between hRad9 and TopBP1 for checkpoint signalling [32], [33]. In Xenopus, human and budding yeast systems TopBP1 has also been shown to directly interact with and activate the ATR-ATRIP protein kinase complex and in Xenopus regulation of this process is 9-1-1 dependent [34], [35], [36], [37]. Recent evidence has demonstrated that chromatin association of TopBP1 following inhibition of DNA replication is reduced in the absence or compromise of Rad17 function and a model for the sequential loading of the checkpoint proteins proposed [38].
Here we have re-examined the role of Rad4TopBP1 in the assembly of pre-replication complexes. Using a chromatin immunoprecipitation (ChIP) assay we report that Rad4TopBP1 associates with origins of DNA replication in a cell cycle-specific manner consistent with a role in assembling a replication complex. We identify a direct protein/protein interaction between Rad4TopBP1 and Mcm10 and demonstrate that association of Mcm10 with origins of DNA replication is dependent on Rad4TopBP1. Although Rad4TopBP1 is present at origins at the replication initiation stage, in contrast to Mcm10, it does not migrate with the replication fork. The recruitment of Sld2 and Sld3 to origins of replication was found to be Rad4TopBP1-dependent. Rad4TopBP1 recruitment at stalled replication forks was also tested and found to be Rad9-dependent and, in contrast to the data cited above [38], Rad17 independent.
Section snippets
Cell growth and media
Cells were grown in YES (0.5% yeast extract, 3% glucose supplemented with 150 mg/l of adenine, leucine, lysine, uracil and histidine) except prior to elutriation where 2% peptone was added to the YES. Hydroxyurea (HU) was added to exponentially growing cells at 10 mM (Table 1).
Elutriation
Centrifugal elutriation of 6 l of exponentially growing cells was carried out using a JE-5.0 (Beckman Coulter) centrifuge. Following elutriation G2 phase cells were grown in YES at either 26 °C or 37 °C. Samples were taken to
Rad4TopBP1 and Mcm10 associate with origins of DNA replication
In order to establish whether Rad4TopBP1 and Mcm10 were associated with origins of DNA replication at the point of initiation we synchronized cells in G2 by incubating cdc25-22 mutants, expressing the relevant functional GFP fusion proteins at the wild type locus, at the restrictive temperature and subsequently releasing them at the permissive temperature to undergo synchronous passage through mitosis, septation and initiation of DNA replication. Control Mcm4-GFP associated with three distinct
Discussion
In budding yeast Mcm10 had previously been shown to associate with origins of DNA replication and the active replisome [18], [20] and our observations extend and confirm these data for S. pombe. If Rad4TopBP1 is to act as a loading factor for Mcm10 at origins of DNA replication then it must associate with those same DNA sequences and this is observed [24] (Fig. 1). Predictably Mcm4 (replicative helicase) and Mcm10 associate with the replisome as it moves outward from the origin of DNA
Conflict of interest
The authors declare there is no conflict of interest.
Funding
This work was supported by Cancer Research UK (M.T., C.P., and J.M.) and the Association for International Cancer Research (K.M. and S.J.A.).
Acknowledgements
We would like to thank Dr. Stephen Kearsey, Prof. Tony Carr and Prof. Hisao Masukata for yeast strains; Adam Watson kindly provided advice for the ChIP procedure and Howard Lindsay and Tony Carr offered comments on the manuscript.
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2013, Molecular CellCitation Excerpt :This in turn couples the checkpoint apparatus to chromatin via interaction of its C-terminal Tudor and BRCT domains with epigenetic modifications on histones H4 and H2A, respectively, in the G1/S phase of the cell cycle (Lin et al., 2012), and facilitates recruitment of the checkpoint kinase, Chk1 (Qu et al., 2012). In a parallel exclusive role in replication, Rad4TopBP1 also facilitates assembly of the Cdc45/Mcm2-7/GINS (CMG) replicative DNA helicase via interaction with Sld3 (Fukuura et al., 2011; Taylor et al., 2011), a function that is conserved in the interaction of TopBP1 with treslin in mammalian systems (Boos et al., 2011; Kumagai et al., 2011). We have now determined the crystal structures of the BRCT1,2 and BRCT3,4 segments of Rad4TopBP1, and structurally and biochemically characterized their phospho-specific interactions with Rad9 and Crb253BP1.
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