GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.
Bottom Line: We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated.Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells.These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.
Affiliation: Genome Stability, London Research Institute, Hertfordshire, UK.
Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.
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Mentions: To initiate DNA replication complexes such as ORC1-6 and the MCM2-7 helicase together with Cdc6 and Cdt1 are loaded onto chromatin to assemble the pre-RC. Origin firing is triggered by Cdk2-CyclinE and Cdc7-Dbf4 kinases that promote the loading of Cdc45 and of polymerases in the presence of Mcm10, TopBP1 and the GINS complex1, 2. To identify novel proteins with a potential role in chromosomal DNA replication in complex organisms we performed database searches looking for open reading frame (ORFs) containing degenerate signature motifs present in known replication factors. We identified an ORF, which we named GEMC1 (GEMinin Coiled-coil containing protein 1), containing a region similar to the coiled-coil domain of geminin, an essential vertebrate replication protein in which the coiled-coil domain is required for its function3 (Fig. 1a and Supplementary Fig 1a). GEMC1 is highly conserved in vertebrates as close homologues can be found in H. sapiens (hGEMC1), M. musculus (mGEMC1), R. norvegicus (rGEMC1) and Xenopus laevis (xGEMC1) (Fig 1b). However, only some of the aminoacid residues critical for geminin function are conserved in GEMC14. In addition, comparison of the predicted GEMC1 structure with geminin revealed an interruption in the coiled-coil domain of GEMC1 (Supplementary Fig 1b). To investigate the role of GEMC1 in DNA replication we isolated xGEMC1 cDNA from Xenopus mRNA and used the Xenopus egg extract system5, 6. We tested whether, similar to geminin, recombinant xGEMC1 was able to inhibit DNA replication3. No significant inhibition of chromosomal DNA replication was observed when physiological amounts of xGEMC1 were added to egg extract (Supplementary Fig 1c) suggesting that xGEMC1’s role is different from geminin. To uncover xGEMC1’s function we generated polyclonal antibodies against recombinant xGEMC1 fusion proteins (Fig. 1c). xGEMC1 is expressed in most Xenopus tissues (Supplementary Fig 1d) with its expression pattern partially overlapping other replication factors such as MCM7, Cdk2 and TopBP1 (data not shown) and is enriched in proliferating cells from skin and gut, although it was also detected in ovary, brain and lung tissues (Supplementary Fig 1d). Analysis in egg extract revealed that xGEMC1 is able to bind chromatin at early stage of DNA replication, although its accumulation progresses more slowly than other replication factors (Fig. 2a). xGEMC1 binding is independent of the MCM2-7 complex as it can be detected on the chromatin in the presence of recombinant geminin, which suppresses MCM2-7 loading without affecting binding of other factors such as TopBP12, 3, 7 (Fig 2a). xGEMC1 loading was minimally affected by Cdk2 inhibitor p272, 3, 7 (Fig 2a). Depletion of Cdc45 did not impair xGEMC1 binding to chromatin although it prevented loading of the GINS complex (data not shown). To investigate xGEMC1 role in DNA replication we depleted xGEMC1 from egg extract (Fig 2b) or supplemented extract with affinity-purified antibodies specific for xGEMC1 to interfere with xGEMC1 function. These treatments inhibited chromosomal DNA replication and did not affect origin independent replication of the single stranded M13 phage (Fig 2c, 2d, 2e and Supplementary Fig 1e). DNA replication inhibition was rescued by the addition of recombinant xGEMC1 to depleted egg extract or by out-competing anti xGEMC1 neutralizing antibodies with an excess of recombinant xGEMC1 (r-xGEMC1) (Fig 2c, 2d and Supplementary Fig 1e). The anti xGEMC1 antibodies did not cross-react with geminin (Supplementary Fig 1f). In addition, nuclear membrane formation, which is required for chromosomal DNA replication8, was not affected by anti xGEMC1 antibodies (Supplementary Fig 2a). xGEMC1 depletion prevented chromatin binding of Cdc45 and Sld5 of the GINS complex and this was restored by recombinant xGEMC1 (Fig 2f). Consistently, anti xGEMC1 neutralising antibodies prevented Cdc45 chromatin loading (Supplementary Fig 2b). TopBP1, Cdc7, ORC1-6 and MCM2-7 complex loading was instead unaffected by xGEMC1 depletion or anti xGEMC1 antibodies (Fig 2f and Supplementary Fig 2c). These data indicate that following its binding to chromatin xGEMC1 is required for Cdc45 and GINS loading onto replication origins. We then performed experiments to identify xGEMC1-binding partners involved in DNA replication. A pull-down with recombinant xGEMC1 fused to Maltose Binding Protein (MBP) and incubated in egg extract co-precipitated Cdc45, CyclinE and Cdk2 (Fig 3a). These interactions were specific as xGEMC1 did not interact with other factors such as Cdc7 and Sld5 (Supplementary Fig 2d). Surprisingly, xGEMC1 was not able to interact with geminin partner Cdt1 (Fig 3a). This is consistent with the fact that aminoacid residues required for Cdt1 binding4 were absent in xGEMC1. We then asked whether any of these interactions were direct using recombinant proteins. We found that recombinant xGEMC1 was able to directly interact with Cdc45 and Cdk2-CyclinE complex in vitro (Supplementary Fig. 3a and 3b). These interactions were also present endogenously as shown by co-precipitation of xGEMC1 with anti Cdc45 antibodies (Supplementary Fig. 3c) and with p13-Suc1 affinity resin for Cdks9 (Supplementary Fig. 3d). We then tested whether xGEMC1 is able to bind TopBP1 (Mus101 in Drosophila, Dpb11 in budding yeast), which is an essential component of the DNA replication machinery2, 7. We found that recombinant xGEMC1 interacts with TopBP1 in extract and in vitro (Fig. 3b). This interaction could also be detected endogenously in xGEMC1 and TopBP1 immunoprecipitates (Supplementary Fig. 3e and 3f).
Affiliation: Genome Stability, London Research Institute, Hertfordshire, UK.