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Manju M. Hingorani


Molecular Biology and Biochemistry




The multi-protein clamp loader complex loads circular clamp proteins onto DNA where they serve as mobile tethers to increase DNA polymerase processivity, and coordinate the functions of many other DNA metabolic proteins. The clamp loading reaction involves multiple steps that occur in two stages: (1) ATP binding enables the clamp loader to bind and open the clamp, and then bind DNA; (2) DNA binding triggers rapid ATP hydrolysis by the clamp loader leading to clamp closure on DNA and complex dissociation. Crystal structures of these proteins show a large number of cationic residues in the clamp and clamp loader that directly contact DNA. In order to investigate the role of these contacts in the clamp loading mechanism, we examined single arginine/lysine mutants of the Saccharomyces cerevisiae clamp, Proliferating Cell Nuclear Antigen (PCNA) and clamp loader, Replication Factor C (RFC) by transient kinetics.

The study of PCNA shows that loss of even a single cationic residue can alter the rates of all DNA-linked steps in the loading reaction, as well as movement of PCNA on DNA. These results explain an earlier finding that each of the nine arginines and lysines in human PCNA is essential for polymerase ฮด processivity. Mutations in the N-terminal domain have greater impact than in the C-terminal domain of PCNA, indicating a positional asymmetry in PCNA-DNA contacts that can influence its functions on DNA.

The study of RFC also shows that removal even one of many cationic residues can alter the rates of DNA-linked steps in the reaction. In addition, we tested the hypothesis that one DNA contact in each RFC subunit can control ATP hydrolysis. Analysis of specific alanine mutants suggest that Arg101 in RFC-D plays a primary role in controlling ATPase activity and Arg88 in RFC-C is important for allowing PCNAโ€ขDNA release to end the clamp loading reaction.



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