Cryo-EM studies of CMG translocation and cohesin loading

Abstract

Before mitosis, the genome must be replicated in the parental cell and sister chromatids must be tethered to ensure accurate division of genes between daughter cells. Within the replisome, DNA unwinding is facilitated by the CMG helicase which contains a hexameric ATPase motor encircling the leading strand template. Intriguingly, the helicase polarity, the mechanism of translocation and how CMG- bound replisome factors such as Mcm10, the MCT complex and Polε affect the helicase has remained elusive. Following replication, sister chromatids are tethered within a dynamic cohesin ring structure. Cohesin loading onto DNA requires ATPase activity and a separate loader complex but it has remained unclear how topological DNA entrapment is mechanistically facilitated or how cohesin engages DNA. In this study I have used cryo-EM together with biochemical assays to investigate how the CMG helicase unwinds DNA and how the cohesin complex is loaded onto DNA. First, I developed a fork affinity purification strategy to establish the binding and translocation polarity of CMG. I then determined the cryo-EM structure of fork-bound CMG, revealing several translocation states with the leading strand template bound to different helicase subunits around the ring. Together with ATPase active site mutations, these structures suggest a rotary but asymmetric translocation mechanism for the helicase. Further analysis of the larger replisome revealed that the MCT complex engages the N-terminal side of the helicase to stabilise parental duplex DNA prior to unwinding. In a collaborative project with Dr Torahiko Higashi, I also determined a cryo-EM structure of the DNA-engaged cohesin complex bound to the loader in a hydrolysis-independent pre-loading conformation. Supported by cross- linking mass spectrometry, FRET and other biochemical assays this structure represents an intermediate state in a two-step cohesin loading mechanism that is completed upon ATP hydrolysis and DNA entry into the cohesin ring.