Structural and Biochemical Studies on the Escherichia Coli Protein MgsA PDF Download

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Languages : en
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In bacterial cells, most if not all replication forks encounter some form of DNA damage or roadblock that stall or inactivate the fork during normal cell growth. Numerous pathways exist for repairing and reactivating replication forks and these pathways are crucial for maintaining genome stability and cell viability. Bacterial "Maintenance of Genome Stability Protein A" (MgsA) and related eukaryotic enzymes are implicated in cellular responses to stalled DNA replication processes. MgsA enzymes are members of the clamp loader clade of AAA+ proteins but their structures and biochemical properties are poorly characterized. We describe the first complete crystal structure of Escherichia coli MgsA that reveals a highly intertwined homotetrameric arrangement for the protein that distinguishes it from other clamp-loader clade AAA+ proteins. An extended oligomerization domain relative to the clamp loader proteins accounts for the unique oligomeric state. The structure represents the inactive conformation of MgsA due to displacement of the arginine finger residues from the neighboring active sites. Thus, a conformational rearrangement is required to engage the arginine finger and activate MgsA ATPase activity. Association with double stranded DNA ends appears to be the trigger that induces the conformational rearrangement and activates ATP hydrolysis. We also describe a potential switch residue, Arginine92, that appears to coordinate DNA binding and ATP hydrolysis within MgsA. MgsA physically interacts with the single-stranded DNA binding protein (SSB). The interaction requires SSB's highly conserved C terminus (SSB Ct) and we define a likely SSB Ct binding site on MgsA. This interaction adds another member to the growing list of SSB interacting proteins and we propose that the interaction is critical for proper MgsA localization to the replisome. Collectively, this thesis presents a structural and biochemical characterization of Escherichia coli MgsA and provides insights into the mechanisms of MgsA-family proteins