Abstract
Human DNA polymerase iota is a low-fidelity template copier that preferentially catalyzes the incorporation of the wobble base G, rather than the Watson-Crick base A, opposite template T (Tissier, A., McDonald, J. P., Frank, E. G., and Woodgate, R. (2000) Genes Dev. 14, 1642-1650; Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and Prakash, L. (2000) Nature 406, 1015-1019; Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Mol. Cell. Biol. 20, 7099-7108). Here, we report on its ability to extend all 12 possible mispairs and 4 correct pairs in different sequence contexts. Extension from both matched and mismatched primer termini is generally most efficient and accurate when A is the next template base. In contrast, extension occurs less efficiently and accurately when T is the target template base. A striking exception occurs during extension of a G:T mispair, where the enzyme switches specificity, "preferring" to make a correct A:T base pair immediately downstream from an originally favored G:T mispair. Polymerase iota generates a variety of single and tandem mispairs with high frequency, implying that it may act as a strong mutator when copying undamaged DNA templates in vivo. Even so, its limited ability to catalyze extension from a relatively stable primer/template containing a "buried" mismatch suggests that polymerase iota-catalyzed errors are confined to short template regions.
Highlights
Human DNA polymerase is a low-fidelity template copier that preferentially catalyzes the incorporation of the wobble base G, rather than the Watson-Crick base A, opposite template T
Tions are Escherichia coli pol1 V, which clearly plays a pivotal role in SOS-induced mutagenesis by copying TT cis-syn dimers, TT (6 – 4) photoproducts and abasic moieties, lesions that typically block normal DNA replication (5–7, 24 –26), and human pol , encoded by POLH ( known as the xeroderma pigmentosum variant (XPV) gene, and/or RAD30A), which plays an important role in the avoidance of sunlight-induced skin cancer by accurately copying cis-syn thymine dimers in vivo [11, 27,28,29]
The Y family DNA polymerases have several important properties in common including low processivity and extremely inaccurate nucleotide incorporation fidelity accompanied by the absence of intrinsic exonucleolytic proofreading in vitro
Summary
Its limited ability to catalyze extension from a relatively stable primer/template containing a “buried” mismatch suggests that polymerase -catalyzed errors are confined to short template regions It has been known for some time that DNA polymerases can be classified into discrete families based upon phylogenetic relationships [1, 2]. The in vitro properties of pol suggest that it might play a role in error-prone translesion replication and possibly in elevated spontaneous mutagenesis, leading to increased carcinogenesis [12, 32] Another potential role for pol is in the somatic hypermutation of human immunoglobulin genes, enabling the production of an efficient immune response to a wide variety of antigens [12, 34, 35]. Sequence-dependent Pol Mismatch Extension results suggest that the efficiency of pol -catalyzed mispair extension is largely dependent upon the template sequence 5Ј to the mispair, and even though pol can extend most mispairs relatively efficiently, a “buried” mispair limits pol synthesis to short regions of DNA
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