A novel ring-like complex ofXenopusproteins essential for the initiation of DNA replication

Abstract

We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…Replication was restored by addition of complete, recombinant GINS, but not by partial complexes. (83) The association of XGINS and XCdc45 with chromatin was sensitive to geminin and to the addition of CDKinhibitor p21. It also depended on the Xenopus homolog of Dpb11.…”

“…(82) The Xenopus homologs of the GINS subunits also form a complex that associates with chromatin at the same time as Cdc45. (83) Depletion of XSld5 from the egg extracts impaired the replication of sperm chromatin. Replication was restored by addition of complete, recombinant GINS, but not by partial complexes.…”

“…It also depended on the Xenopus homolog of Dpb11. (83,84) Interestingly, electron microscopy of purified recombinant GINS proteins revealed a ring-shaped structure. (83) The GINS ring pores are approximately 4 nm in diameter, sufficient to encircle DNA, but their function is still unknown.…”

Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1

“…The lack of Rad17 dependency does however contrast with previous work in Xenopus demonstrating a strict requirement for Rad17 to effect TopBP1 chromatin loading following checkpoint activation (38,52). One possible explanation for this dichotomy might be the fact that there are additional Rfc1-like proteins in yeast (53,54) and the 17 observed Rad17 independence might then reflect functional redundancy in this organism, but this remains to be tested. A second possibility might lie in the fact that the Xenopus assay relies on bulk chromatin association whereas here TopBP1 association with mapped stalled replication forks is assessed.…”

“…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).…”