Electron microscopic analysis reveals that replication factor C is sequestered by single-stranded DNA

Replication factor C (RF-C) is a eukaryotic heteropentameric protein requir ed for DNA replication and repair processes by loading proliferating cell n uclear antigen (PCNA) onto DNA in an ATP-dependent manner, Prior to loading PCNA, RF-C binds to DNA, This binding is thought to be restricted to a spe cific DNA structure, namely to a primer/template junction, Using the electr on microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The electron microscopic dat a indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA, No preferential a ffinity for the 3' or 5' transition points from single- to double-stranded DNA was evident.