Dpb11 may function with RPA and DNA to initiate DNA replication.

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication.

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described.

Origin DNA Melting-An Essential Process with Divergent Mechanisms.

Origin DNA melting is an essential process in the various domains of life. The replication fork helicase unwinds DNA ahead of the replication fork, providing single-stranded DNA templates for the replicative polymerases. The replication fork helicase is a ring shaped-assembly that unwinds DNA by a steric exclusion mechanism in most DNA replication systems. While one strand of DNA passes through the central channel of the helicase ring, the second DNA strand is excluded from the central channel. Thus, the origin, or initiation site for DNA replication, must melt during the initiation of DNA replication to allow for the helicase to surround a single-DNA strand. While this process is largely understood for bacteria and eukaryotic viruses, less is known about how origin DNA is melted at eukaryotic cellular origins. This review describes the current state of knowledge of how genomic DNA is melted at a replication origin in bacteria and eukaryotes. We propose that although the process of origin melting is essential for the various domains of life, the mechanism for origin melting may be quite different among the different DNA replication initiation systems.