Coordination and Regulation of Msh2-Msh3 Functions During DNA Metabolism
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Eukaryotic DNA replication is a tightly regulated process where the genetic material of a cell is faithfully copied and transferred to its offspring. The fidelity of this process is constantly challenged by internal or external insults to the DNA, such as chemical agents or by-products of metabolism. If the damage is left unrepaired, errors in the DNA can have a positive or negative impact, e.i., mutations can generate evolutionary diversity but can also play a role in the development of disease. To guarantee stable genome inheritance, organisms developed a myriad of sophisticated strategies to prevent inaccurate synthesis, and repair mechanisms to correct the occasional errors that are formed. Mismatch repair (MMR) is an evolutionarily conserved repair pathway essential to safeguard the genome from errors that occur during genetic recombination and DNA replication. In eukaryotes, two MutS homologs (Msh) initiate MMR, either Msh2-Msh3 or Msh2-Msh6. These heterodimer complexes recognize misincorporated nucleotides or mismatches with different efficiencies. While Msh2-Msh6 recognizes base-base misinsertions and small insertion-deletion loops (IDLs), Msh2-Msh3 preferentially binds to large IDLs. The role of Msh complexes is not limited to correcting post-replicative DNA lesions. For instance, Msh2-Msh3 also interacts with a wide range of DNA lesions that include branched intermediates with 3’ or 5’ single-strand/double strand (ss/ds) DNA flaps. Interestingly, while the binding to 3’ single- strand (ss) DNA leads to repair in a specialized double strand repair pathway via a 3’ non-homologous tail removal (3’NHTR), in vitro evidence suggest that Msh2-Msh3 binding to 5’ flap structures not only interferes with the normal processing of the intermediate by Fen1 (Rad27) and DNA Ligase 1 (Cdc9), but it is the mediator of introducing small expansions in a TNR sequence context. Trinucleotide repeat (TNR) expansions is the underlying cause for over forty neurodegenerative and neuromuscular diseases in humans.This work is focused on understanding the interplay between Msh2-Msh3 interactions with specific substrates and how these interactions lead to genome stability or instability outcomes. I first set out to understand how Msh2-Msh3 facilitates 3’NHTR. Despite the known interactions and biochemical activities among Saw1, Msh2-Msh3, RPA, Slx4 and Rad1-Rad10, the mechanistic role(s) that each protein plays in 3’NHTR remains unclear. How are these proteins coordinated to allow 3’NHTR? To address this question, a combination of genetic and biochemical approaches were used to study 3’NHTR in Saccharomyces cerevisiae. In Chapter 2 I discuss our findings and provide a cohesive model of how these proteins are coordinated for efficient 3’ NHTR. In Chapter 3 the focus shifts to determining the previously uncharacterized in vivo consequences of Msh2-Msh3 binding to 5’ flap intermediates. I show that overexpression of Msh2-Msh3, but not Msh2-Msh6, interferes with normal cell cycle progression, specifically DNA replication, potentially inducing Okazaki fragment processing stress. Notably, this effect is dependent on Msh2-Msh3 ATPase activity, as well as downstream MMR factors. This observation is consistent with TNR models that suggest Msh2-Msh3’s abundance and ATPase activity are required to drive genome instability.