Probing the Active Sites of Rat DNA Polymerase Beta and Human Topoisomerase I

We studied the function of key amino acids in DNA polymerase beta and topoisomerase I. The methodology employed misacylated tRNAs to incorporate alternate amino acids at predetermined positions. The first enzyme, DNA polymerase beta, plays a crucial role in the base excision repair pathway. The base excision repair pathway is responsible for removal of 10,000 lesions per cell per day that result from oxidative and alkylation damage. Environmentallymediated DNA damage poses a daily threat to the survival of eukaryotic cells. This damage, when left unrepaired, can result in mutations. The second enzyme, human topoisomerase I (htopo1), relieves the torsional strain in DNA that is built up during replication and transcription. It is vital for cell proliferation and is the target for poisoning by anticancer drugs. Misacylated suppressor tRNAs were employed to insert single, non-natural amino acids into both rat DNA polymerase beta and htopo1. The resulting modified enzymes were then compared to the wild-type enzymes to study the effect of the modifications. The data showed that the modified polymerases beta had reduced catalytic abilities and, interestingly, one mutant exhibited differential sensitivity to an inhibitor. The data also showed that htopo1 is sensitive to modification at position 533, but not sensitive to modification at position 356.

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