Our long term objectives are to understand the roles of human DNA polymerases, Pol-eta, Pol-iota, and Pol-kappa, in the replication of damaged DNA.
In Specific Aim 1, it is proposed that for replication to occur through highly distorting DNA lesions, two different DNA polymerases are required, wherein Pol-iota carries out nucleotide incorporation opposite the lesion and Pol-kappa extends from the inserted nucleotide. By contrast, Pol-eta is suggested to replicate only through moderately distorting lesions. These ideas will be tested using propano adducts that are highly distorting and affect Watson-Crick base pairing, and which have been shown to be present at high levels in human DNA. Propano adducts are formed in DNA from the reaction of the reactive N2 of guanine with alpha,beta-unsaturated aldehydes or enals, which include acrolein and crotonaldehyde, that are ubiquitious pollutants in the environment and which are also formed in cells as the end product of peroxidation of lipids, and trans-4-hydroxy-2-nonenal, a unique oxidation product of omega-6 polyunsaturated fatty acids.
In Specific Aim 2, it is proposed that Pol-kappa can efficiently extend mispaired primer termini either by incorporating the next correct nucleotide or by using a specific primer-template misalignment mechanism that generates -1 deletions. The proficiency of Pol-kappa to extend mispaired primer termini by these two means will be examined by steady-state kinetic analyses.
In Specific Aim 3, it is proposed that because of their proficient ability to extend from mispaired primer termini, both Pol-kappa and Pol-zeta would contribute to mutagenesis in undamaged as well as UV damaged cells in humans. This idea will be tested by carrying out genetic studies in the fission yeast S. pombe to examine the relative contributions of Pol-kappa and Pol-zeta to spontaneous and UV induced mutagenesis.
In Specific Aim 4, the manner by which the switch from the replicative polymerase, Pol-delta, to a translesion synthesis polymerase occurs at the lesion site will be studied and the role of PCNA as the key intermediary in this process delineated. Our understanding of the manner in which human cells overcome blocks to DNA replication should be greatly enhanced by these studies.
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