Key steps in the loading and activation of the replicative helicase MCM2-7

Abstract

The eukaryotic cell cycle coordinates duplication of the genome and segregation of the DNA to maintain genomic stability. Each chromosome is replicated from multiple DNA replication origins. Only after the completion of S-phase cells segregate their chromosomes in two daughter cells. To maintain genomic stability each origin is activated only once, which is achieved by a two-step mechanism. The first step, called DNA licensing, involves the loading of MCM2-7 helicase into pre-Replication Complexes (pre-RC) at replication origins. In the second step, the helicase becomes activated by S-phase kinases and several replication factors to form replication forks that replicate the DNA. The reconstitution of helicase loading and its structural characterisation by cryo-EM has generated unprecedented insights into pre-RC formation.  Here, the six-subunit origin recognition complex (ORC), Cdc6 and Cdt1 cooperate to load two MCM2-7 hexamers into a salt-stable double-hexamer around double-stranded DNA. During the seminar I will present biochemical and structural insights into this multi-step assembly process, detailing how initial complex formation occurs and how MCM2-7 ring closure around DNA coordinates quality control of complex assembly. I will show the structure of the MCM2-7 double-hexamer encircling double-stranded DNA, which together with biochemical data reveals how the complex is kept inactive in G1 phase, but is primed for helicase activation during S-phase. In summary, the mechanistic and structural discoveries over the last 10 years start to explain how DNA replication is initiated in Saccharomyces cerevisiae.