Fidelity of Human DNA Polymerase η

Abstract

Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol. Of the eukaryotic DNA polymerases, only human Pol and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol misincorporates nucleotides with a frequency of ϳ10 ؊2-10 ؊3, and importantly, hPol synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.The Saccharomyces cerevisiae RAD30 gene is involved in error-free bypass of UV-damaged DNA (1-3). RAD30 encodes a DNA polymerase, Pol, that can efficiently replicate DNA past a cis-syn thymine-thymine (T-T) 1 dimer in template DNA, and it does so by inserting two adenines across from the two thymines of the dimer. (4) Cells from the cancer-prone, variant form of xeroderma pigmentosum (XP-V) are much slower than normal cells in replicating DNA containing UV damage (5, 6), and XP-V cell-free extracts are deficient in replicating DNA past a cis-syn T-T dimer (7). The hRAD30A gene encodes the human counterpart of yeast RAD30, and mutations in hRAD30A cause XP-V (8, 9). Human Pol, like it's yeast counterpart, bypasses a cis-syn T-T dimer (9). Here, we determine the fidelity of hPol opposite all four nondamaged template bases, as well as opposite the T-T dimer, by measuring the steady state kinetics of deoxynucleotide incorporation. We find that hPol is a low fidelity DNA polymerase. Interestingly, hPol misincorporates deoxynucleotides opposite the T-T dimer with the same frequency as it does opposite the nondamaged template bases, ϳ10 Ϫ2 -10 Ϫ3 . These observations suggest that hPol has a flexible active site that is relatively insensitive to geometric distortions in DNA and that this property enables the enzyme to replicate past a T-T dimer. MATERIALS AND METHODSOverexpression of the hRAD30A Gene-To overexpress the human Pol protein (hPol), the hRAD30A gene was cloned in-frame with the glutathione S-transferase gene, which is under the control of a galactose-inducible phosphoglycerate promoter. The hRAD30A gene was amplified from the genomic clone GS21749 (8) by PCR using the primer oligonucleotides 5Ј-CCCTGAAGCTTGGATCCACATATGGCTACTGG-ACAGGATCGAGTGGTTGCTC-3Ј and 5Ј-CAGGGAATTCGGATCCAA-TATTAAATCCTACAGGCAAGCCTGAGGGCAGC-3Ј, which generate BamHI restriction sites 6 nucleotides upstream and 35 nucleotides downstream of the hRAD30A open reading frame, respectively. The wild type 1.3-kilobase pair SnaBI/NcoI DNA fragment from GS21749, which contains an internal portion of hRAD30A, was t...

8-(Hydroxymethyl)-3,N4-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases

“…Pol η also replicated the unmodified template, forming all four base substitutions (Figs 3 and 4). This highly error-prone nucleotide incorporation supports previous findings that the enzyme replicates normal DNA with low fidelity (45)(46)(47). Some of this may be explained by a loose requirement of the polymerase for correct Watson-Crick base-pairing geometry.…”

UV-B Radiation Induces Epithelial Tumors in Mice Lacking DNA Polymerase η and Mesenchymal Tumors in Mice Deficient for DNA Polymerase ι

“…Thus, two-thirds and one-third of UV-induced CPDs in cells are thymine-thymine and thymine-cytosine photoproducts, respectively. On the other hand, it was clearly shown that thymine-thymine CPDs are largely bypassed in an accurate manner by human Pol (28,41,42). Hence, inactivation of Pol renders cells hypermutable and mice susceptible to cancer by UV irradiation.…”

Resposta a danos no DNA após exposição à luz ultravioleta: apagando o fogo antes do incêndio celular

“…Dois modelos explicam como isso poderia acontecer: A inserção sempre de adenina oposta a um dímero por polimerases síntese translesão ou a replicação de uracila, resultado da desaminação da citosina em um dímero de pirimidina. comparado às polimerases clássicas da família B (McCulloch e Kunkel, 2008), entretanto, dependendo da polimerase TLS recrutada, a lesão pode ser replicada praticamente sem erro como Pol η tendo CPD de timinas (T^T) como molde (Johnson et al, 2000). A outra lesão gerada por luz UV, o díme-ro 6-4PP, possui uma distorção muito maior e não pode ser replicada por Pol η e estudos indicam que Pol ζ e REV1 são necessárias para transpor essa lesão, porém com maior taxa de mutagênese (Nakajima et al, 2004).…”