Interplay Between Mcm Helicase And Checkpoint Proteins Rescues Replication Fork Defects Of Mcm10

Mcm10 is essential in the initiation and elongation of DNA replication. It is implicated in the activation and stable assembly of various elongation factors such as the MCM2-7 helicase, Cdc45, and polymerase alpha primase (Pol-alpha) at the replication fork based on its physical interactions with these proteins. Second site suppressors of two temperature labile mcm10 mutants have been identified and they have been shown to cluster in two regions of Mcm2 located at the interface of adjacent subunits of the hexameric MCM helicase. These dominant mcm2 suppressors restore viability to the mcm10 mutants without restoring the stability of Mcm10p, the interaction of Mcm10 with Mcm2, or the replication initiation defects of mcm10. Rather, they alleviate the elongation defect of mcm10 in that they suppress the HU and MMS sensitivity and the fork pausing phenotype of mcm10 as well as restore stability of Pol-alpha. This suppression requires the activity of genes involved in replication fork restart as well as key checkpoint regulators such as Rad53 and Mec1. Furthermore, stabilization of Polalpha is dependent on Mec1. These results suggest that at the restrictive temperature mcm10 causes destabilization of the replication fork that result in degradation of Pol-alpha. This fork defect is alleviated by the altered activity of the MCM helicase as well as the coordinated action of checkpoint proteins that stabilize replication forks