Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Florida State University College of Medicine, United States.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2013; 288(11). DOI: 10.1074/jbc.M112.440941
Source: PubMed


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.

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    • "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. "
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