Fidelity and Processivity of Saccharomyces cerevisiae DNA Polymerase η

Abstract

The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol , that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steadystate kinetic analyses of deoxynucleotide incorporation indicate that pol has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10 ؊2 to 10 ؊3 . Also pol has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol 's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.Mutations in the RAD30 gene of Saccharomyces cerevisiae confer moderate sensitivity to ultraviolet (UV) radiation, and genetic studies have indicated the involvement of this gene in error-free bypass of UV-damaged DNA (1-3). RAD30 encodes a DNA polymerase, pol , 1 that can efficiently and correctly bypass a cis-syn-thymine-thymine (T-T) dimer in template DNA by inserting two adenines opposite the two thymines of the dimer (4). The Rad30 DNA polymerase activity is essential for resistance to UV radiation and for its role in error-free bypass (3). Recently, the human homologue of pol has been identified, and mutational alterations in this protein are responsible for the variant form of the cancer-prone genetic disorder xeroderma pigmentosum (XP-V) (5, 6).Most DNA polymerases misincorporate deoxynucleotides with very low frequencies, in part because they prefer to insert deoxynucleotides that form correct Watson-Crick base pairs and which do not distort the Watson-Crick geometry (7). Thus, even though the capacity for base pairing recognition is retained in the cyclobutane pyrimidine dimer (8, 9), a T-T dimer is still a block to most DNA polymerases, including the eukaryotic replicative polymerase pol ␦ (4), presumably because of the intolerance of their active site to DNA distortion caused by the dimer (10, 11). In contrast, the ability of pol for efficient and correct bypass of a T-T dimer may derive from an unusual active site that is more tolerant of DNA distortions. In that case, pol would be expected to have a low fidelity (see also the "Discussion"). Here we determine the fidelity of pol by measuring the steady-state kinetics of correct and incorrect deoxynucleotide incorporation and also examine its processivity. We find that pol is a low fidelity polymerase, and it incorporates only a few nucleotides before dissociating from the primer-template DNA substrate. This low processivity may restrict pol synthesis to short patches, thereby minimizing the error frequency, and thus accounting for its role in error-free bypass of UV-damaged DNA. MATERIALS AND METHODSDNA Substrates-The following four (53-mer) oligodeoxynucleotides were used as templates, and they differ only in the underlined sequences: Template G, 5Ј-ATGC...

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