Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying
this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly
to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction
between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited, and excess RPA accumulates
at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement
for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase
during replication stress.
"Although current data are consistent with such a model, alternative and/or additional scenarios are possible. In particular, Cdc45 may mediate stalling and disruption of CMG on replication stress, as proposed recently (55).Because Cdc45 is essential for an active replicative helicase and is used to start origins of replication, it is tempting to speculate that once a bubble has been opened, e.g. by a spiral conformation of the Mcm2-7 complex, Cdc45 may act as pathfinder to define the ss/ds junction and keep it open until the spiral form of Mcm2-7 rearranges to the ring-conformation encircling the leading strand. "
[Show abstract][Hide abstract] ABSTRACT: The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.
Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1217 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genome duplication requires the coordinated action of multiple proteins to ensure a fast replication with high fidelity. These factors form a complex called the Replisome, which is assembled onto the DNA duplex to promote its unwinding and to catalyze the polymerization of two new strands. Key constituents of the Replisome are the Cdc45-Mcm2-7-GINS helicase and the And1-Claspin-Tipin-Tim1 complex, which coordinate DNA unwinding with polymerase alpha-, delta-, and epsilon- dependent DNA polymerization. These factors encounter numerous obstacles, such as endogenous DNA lesions leading to template breakage and complex structures arising from intrinsic features of specific DNA sequences. To overcome these roadblocks, homologous recombination DNA repair factors, such as Rad51 and the Mre11-Rad50-Nbs1 complex, are required to ensure complete and faithful replication. Consistent with this notion, many of the genes involved in this process result in lethal phenotypes when inactivated in organisms with complex and large genomes. Here, we summarize the architectural and functional properties of the Replisome and propose a unified view of DNA replication and repair processes.
[Show abstract][Hide abstract] ABSTRACT: The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays an important role in the intra-S-phase checkpoint. Its interactions with other replication proteins during the cell cycle and after intra-S-phase checkpoint activation are only partially characterized. Here, we present that the C terminal part of Cdc45 may mediate its interactions with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A (RPA) and DNA polymerase δ are maximal during S phase. Following UVC-induced DNA damage, Cdc45-Claspin complex formation is reduced whereas the binding of Cdc45 to RPA is not affected. We also show that treatment of cells with UCN-01 and Phosphatidylinisitol 3-kinase-like kinase inhibitors does not rescue the UV-induced destabilisation of Cdc45-Claspin interactions, suggesting that the loss of interaction between Cdc45 and Claspin occurs upstream of ATR activation in the intra-S-phase checkpoint. This article is protected by copyright. All rights reserved.
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