Jerard Hurwitz: Studies with Alternative Clamp Loader Derivatives

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The five subunit complex RFC loads PCNA onto DNA and is essential for replication. Recently, multiple derivatives of RFC (the replicative clamp loader) have been isolated and shown to play specific roles in DNA transactions. A five subunit complex in which the large subunit of RFC (RFC1) is replaced by Rad17 (Rad17-RFC) is involved in DNA damage response and replication check-point control. Rad17-RFC specifically loads a three subunit complex Rad9-Hus1-Rad1 onto damaged DNA. Another RFC derivative, Ctf18-RFC, in which the subunit RFC-1 is replaced by a complex of Ctf18-Ctf8-Dcc1, is involved in cohesion. Cohesion is the process by which sister chromatids are bound together from the time they are duplicated in S-phase until the DNA is ready to be distributed equally between daughter cells during anaphase. Genetic studies, primarily in yeasts, have identified a number of genes essential for sister-chromatid cohesion which are categorized into three functional groups: structural proteins, deposition factors and establishment factors. The structural factors include the cohesin subunits, Smc1, Smc3, Scc1 and Scc3 which form a circular four subunit structure. Sister chromatids are held together by cohesin after DNA replication. These cohesin connection are severed through the cleavage of the Scc1 subunit by separase during cell division. The deposition factors include Scc2 and Scc4 which bind to the Pre-RC prior to the initiation of replication and load cohesin on chromatin. The establishment factors include Ctf7, Ctf4, Ctf18, Dcc1, Ctf8 and CHlR1 that function during S-phase and interact both directly and genetically with replication proteins. The temporal requirement for the function of cohesion establishment factors and their interactions suggest that sister chromatid cohesion is coupled to DNA replication. The conservation of the genes involved in cohesion from yeast to human indicate that the overall process is evolutionally conserved. Our studies have revealed that the cohesion Ctf18-RFC, like the replicative RFC, loads PCNA onto DNA. We have detected direct interactions between Ctf18-RFC and a number of the cohesion proteins including the four-subunit cohesin complex, Ctf7, Ctf4 and CHlR1. Both Ctf7 and Ctf4 interact with PCNA while CHlR1 contains 5’ to 3’ DNA helicase activity, suggesting that the establishment factors play a role associated with DNA transactions. Preliminary data indicate interactions between DNA polymerase e and Ctf18-RFC. Our present studies are focused on how the establishment factors, in conjunction with DNA polymerase e, interact to modify naked DNA as well as chromatinized DNA loaded with cohesin. Our present model of sister chromatid cohesion, shown in the figure below, is that there is a remodeling of the proteins at the replication fork that permits the fork to pass through sites at which the cohesin complex is deposited.