Mechanisms of Helicases, Translocases and SSB Proteins involved in Genome Maintenance

Washington University, Saint Louis MO

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Abstract

Abstract We are studying two classes of DNA binding proteins, DNA helicases and single stranded (ss)DNA binding (SS8) proteins, both of which are essential for genome maintenance in all organisms. DNA helicases are ATP-dependent molecular motors that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA replication, recombination and repair. SS8 proteins bind tightly to these ssDNA intermediates, protecting the DNA, but also bind directly to at least 20 other SS8 interacting proteins (SIPs) to bring them to their sites of action. Defects in DNA helicases are responsible for a number of human diseases. We are undertaking quantitative studies of the mechanisms of DNA unwinding and ssDNA translocation of several superfamily 1, non-hexameric DNA helicases. E. coli Rec8CD functions in repair of DNA double strand breaks, E. coli UvrD and Rep helicases which function in several DNA repair pathways and M. tuberculosis (Mt) UvrD1 helicase. Rec8CD is a hetero-trimeric complex containing two superfamily 1 (SF1) helicase/translocase motors (Rec8, a 3' to 5' motor and RecD, a 5' to 3' motor). We have discovered that Rec8CD can unwind duplex DNA processively even in the absence of ssDNA translocation by the canonical Rec8 and RecD motors and that DNA melting and ssDNA translocation are separate processes, with DNA melting and DNA unwinding rates regulated by its nuclease domain. E. coli UvrD, Rep and Mt UvrD1 monomers are rapid ssDNA translocases, but do not possess processive helicase activity due to autoinhibition by a 28 sub-domain. Helicase activity requires activation by dimerization in the absence of accessory proteins. We have shown that the Mt UvrD1 dimerization involves a Cys-Cys disulfide bond between the 28 sub-domains of the two subunits and dimerization is under redox control. We have determined Cryo-EM structures of the UvrD1 dimer bound to a DNA substrate, the first such structures of an SF1 dimer. How this dimer translocates and unwinds DNA will be a focus of our studies. Rep and UvrD monomers can also be activated through interactions with the accessory proteins, PriC and MutL, respectively. Despite extensive study, the mechanism of helicase initiation and DNA unwinding is not well understood for SF1 helicases. We will determine what is needed to turn a ssDNA translocase into a helicase and how this is regulated. E. coli SS8 protein is a central player in all aspects of DNA metabolism. It can bind ssDNA in multiple binding modes that differ dramatically in their properties, in particular ssDNA binding cooperativity. We have shown that the intrinsically disordered C-terminal tails of SS8 regulate cooperative binding of SS8 to ssDNA as well as phase separation of SS8. We have shown that glutamate promotes both cooperative binding to DNA as well as phase separation of SS8 in the absence of DNA. We propose CryoEM studies to examine SS8-DNA cooperativity and the role of the C-terminal tails. An array of approaches, including thermodynamic, transient kinetic, Cryo-EM and single molecule approaches, will be used in these studies.