Abstract
DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Pol eta bypasses this lesion extremely poorly. These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation.
Highlights
The yeast RAD30 gene, which belongs to the RAD6 epistasis group, encodes a DNA polymerase, Pol, that has the unique ability to efficiently replicate through a cis-syn thymine-thymine (T-T) dimer; it does so correctly by inserting two A resi
To further assess the range of DNA lesions tolerated by Pol, here we examine the bypass of an abasic site, a prototypical noninstructional lesion
To further assess the range of template lesions tolerated by Pol, here we examine the bypass of an abasic site, a prototypical noninstructional lesion
Summary
The yeast RAD30 gene, which belongs to the RAD6 epistasis group, encodes a DNA polymerase, Pol, that has the unique ability to efficiently replicate through a cis-syn thymine-thymine (T-T) dimer; it does so correctly by inserting two A resi-. Most DNA polymerases are highly sensitive to geometric distortions in DNA [20], and their fidelity is affected more severely by the disruption of optimal geometry than by H bonding between base pairs [21, 22]. As a consequence, they are unable to incorporate nucleotides opposite lesions that distort the DNA helix. We suggested that the ability of Pol to bypass lesions, such as the T-T dimer, 8-oxoG, and m6G, results from an unusual tolerance of its active site for the distorted template geometries of these lesions. These results suggest that Pol requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide insertion
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