Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase ι

Humans possess four Y-family polymerases: pols eta, iota, kappa and the Rev1 protein. The pivotal role that poleta plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding poleta), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols iota, kappa and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of poliota to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that poliota can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, poliota efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of similar to10(2) over the incorporation of incorrect nucleotides, while incorporation opposite the 51 is highly mutagenic. Polkappa has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions Of poliota and polkappa result in a significant increase in bypass of T-T CPDs when compared to poliota alone. Our data suggest that under certain conditions and sequence contexts, poliota, can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochemical activities may, therefore, be of biological significance especially in XP-V cells lacking the primary T-T CPD bypassing enzyme, poleta. (C) 2003 Elsevier B.V. All rights reserved.