Linchpin DNA-binding residues serve as go/no-go controls in the replication factor C-catalyzed clamp-loading mechanism.

摘要: DNA polymerases depend on circular sliding clamps for processive replication. Clamps must be loaded onto primer–template (ptDNA) by clamp loaders that open and close around ptDNA in an ATP-fueled reaction. All share a core structure which five subunits form spiral chamber binds the at its base twisted encloses within, while binding hydrolyzing ATP to topologically link ptDNA. To understand how perform this complex task, here we focused conserved arginines might play central coordinating role mechanism because they can alternately contact or Walker B glutamate ATPase site lie loader–clamp-binding interface. We mutated Arg-84, Arg-88, Arg-101 ATPase-active B, C, D of Saccharomyces cerevisiae replication factor C (RFC) loader, respectively, assessed impact multiple transient events reaction: proliferating cell nuclear antigen (PCNA) binding/opening/closure/release, binding/release, hydrolysis/product release. The results show these relay critical information between PCNA-binding, DNA-binding, sites all steps reaction, particularly checkpoint before RFC commits hydrolysis. Moreover, their actions are subunit-specific with RFC-C Arg-88 serving as accelerator enables rapid hydrolysis upon RFC-D brake confers specificity correct substrate loading PCNA.