Based on these results, we conclude the BPII region of Claspin plays an important role in the binding of Claspin to branched, replication fork-like DNA structures. Open in a separate window Figure 5 The Claspin N terminus, but not the C terminus, binds to replication fork-like DNA in G007-LK vitro. may be important for Claspin function during DNA replication and DNA replication checkpoint signaling. strain lacking Mrc1, the replicative MCM (minichromosome maintenance) helicase co-factor Cdc45 becomes actually uncoupled from the sites of DNA synthesis,18 and the leading strand polymerase Pol (DNA polymerase epsilon) becomes destabilized at stalled replication forks.20 Though much of this initial work focused on exogenous agents that induce replication stress, it has since been discovered that repetitive DNA sequence elements and alternative DNA structures are unstable and sensitive to breakage during DNA replication in candida strains lacking Mrc1.21C24 Observations that Mrc1 immunoprecipitates with several DNA replication fork-associated proteins, including Cdc45 and Pol ,18,20,25C27 led to the suggestion that Mrc1 physically couples the activities of the replicative helicases and polymerases during DNA G007-LK replication to prevent genome destabilization. Studies using Xenopus egg components have also isolated protein complexes comprising Claspin with these and additional replication-fork associated factors,28 therefore indicating that the part of Mrc1 in DNA replication may be conserved through development. Recent studies in human being cells have similarly suggested that human being Claspin is required for normal rates of replication fork progression29,30 and to prevent genome rearrangements that may arise at stalled replication forks.31 How Claspin associates with replication forks and regulates replication fork TRADD progression is currently unclear. Though Claspin and its fission candida homolog Mrc1 both preferentially bind to replication fork-like DNA constructions in vitro,32,33 Claspin does not stably associate with chromatin in the absence of Cdc45.34,35 Since Cdc45 is required for the replicative MCM helicase to unwind DNA during DNA replication,36C38 these results suggest that the association of Claspin with replication forks may be dependent on the replication fork structures that are generated from the MCM helicase and/or on direct protein-protein interactions with Cdc45. Furthermore, the Timeless-Tipin complex (budding candida Tof1-Csm3 and fission candida Swi1-Swi3) is an additional protein factor that associates and techniques with replication forks inside a Cdc45-dependent manner.18,35 The removal of Timeless-Tipin from Xenopus egg extracts and its deletion in yeast also helps prevent the association of Claspin and Mrc1 with replicating chromatin.26,35,39 Taken together, these effects suggest that several protein-protein and protein-DNA interactions may be important for Claspin recruitment and function at replication forks during DNA replication and in replication checkpoint signaling. To identify the specific protein-protein and protein-DNA relationships that may be important for Claspin function, we purified human being Claspin, several of its practical domains and a number of replication fork-associated proteins that have been shown to co-immunoprecipitate with Claspin. We then characterized the binding of the Claspin fragments to DNA polymerase epsilon, Rad17-RFC, Cdc45, Timeless and branched, replication fork-like DNA. Our results show the Claspin N terminus binds to Cdc45, Timeless and DNA, and that the C terminus binds to DNA polymerase epsilon and Rad17-RFC. These results suggest that the binding of different regions of Claspin to specific proteins and to DNA may be important for its function in DNA replication and replication checkpoint signaling. Results Claspin interacts with several replication fork-associated proteins in nuclear components. Co-immunoprecipitation studies of Xenopus Claspin and budding and fission candida Mrc1 have indicated that several replication fork-associated proteins interact with Claspin and Mrc1, including DNA polymerase epsilon (Pol ), Cdc45, Rad17-RFC and the Timeless-Tipin complex.18,20,25C28,39C41 To determine whether any of these interactions are conserved in human Claspin, we immunoprecipitated endogenous Claspin from HeLa nuclear extract using an antibody raised against an internal segment of G007-LK human Claspin (amino acids 725C775). As demonstrated in Number 1A, we observed the presence of the catalytic subunit of Pol , the DNA damage/replication checkpoint protein Rad17 and the MCM helicase co-factor Cdc45 in the Claspin immunoprecipitates, but G007-LK not in the control IgG immunoprecipitates. We note that at an association of Claspin with the human being homologs of Pol and Cdc45 has not been previously reported, though it was expected based on the evolutionary conservation of these proteins. Rad17 was previously shown to co-immunoprecipitate with human being Claspin41 and thus served like a positive control with this experiment. Furthermore, we did not observe significant amounts of the replicative clamp protein PCNA in complex with Claspin, suggesting the isolated Claspin immunocomplexes were specific and unique. G007-LK Though the replication fork-associating Timeless-Tipin complex has been shown to co-immunoprecipitate with Claspin,35,39,42,43 its absence in these particular immunocomplexes indicates the connection of Claspin with Pol , Rad17-RFC and Cdc45 happen self-employed of Timeless-Tipin. We consequently conclude the Claspin-Cdc45/Pol /Rad17 complexes are specific and self-employed of other proteins that will also be present at replication forks. Open in a separate window Figure.