IN VITRO ANALYSIS OF A DNA REPLICATION APPARATUS

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DNA, being the genetic repository of the cell, must be duplicated and maintained with very high accuracy. A combination of 7 purified replication proteins of phage T4 is able to copy DNA in vitro with an accuracy comparable to that observed during DNA replication in vivo. We plan to analyze the mechanisms involved in control of error rates by neighboring DNA sequence using this system. In particular, we shall determine (a) the number of base pairs in the neighboring sequence that are perturbed due to T:G, T:C, T:T, A:A, A:C, A:G, G:A and G:G mispairs, (b)\any relationships between the thermal stability of the neighboring DNA sequence and the degree of observed perturbation and (c) which proteins contribute to the recognition of the perturbation due to a mispair. A variety of agents in our environment constantly change the structure and informational content of DNA in our cells. There is a large battery of repair mechanisms that monitor such alterations and correct them. Nevertheless, some of these alterations escape repair and lead to mutations and cancer. So far, attempts to understand the mechanisms of mutagenesis due to carcinogens have utilized enzymes of relatively low accuracy as probes. We plan to use the highly accurate replication complex of phage T4 as a probe. Modified deoxynucleotides resulting from carcinogen interaction with the normal dNTP substrates of DNA will be synthesized and purified. The pairing properties of these altered dNTPs will be examined using sensitive sequencing and infectivity assays. DNA templates containing these modified nucleotides in specific sites in natural DNA will be constructed. The pairing properties of these modified nucleotides as template residues during DNA replication will be examined both in vitro and in vivo.