Imaging analysis to determine chromatin binding of the licensing factor MCM2-7 in mammalian cells.

Abstract

S-CDK and DDK protein kinases initiate DNA replication at replication origins. Prior to the activation of these kinases, origins must become competent for replication by loading MCM2-7 DNA helicase on chromatin. This process is known as replication licensing or pre-replicative complex (pre-RC) formation. After the onset of S phase, however, licensing is inhibited to prevent re-replication of DNA. In this chapter, we describe a method to analyze origin licensing by imaging the chromatin-bound licensing factor MCM2-7. In a normal cell cycle, MCM2-7 is loaded at the end of mitosis or early G1 phase. As S phase progresses, MCM2-7 is dissociated from the replicated regions. When DNA replication is completed, cells in G2 phase have no chromatin-bound MCM2-7. The analysis of chromatin-bound MCM2-7 in each cell provides an insight into cell cycle stage and condition for cell cycle.