A unique error signature for human DNA polymerase nu.
Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.
Arana
Mercedes E
ME
Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.
Takata
Kei-ichi
K
Garcia-Diaz
Miguel
M
Wood
Richard D
RD
Kunkel
Thomas A
TA
D000595:Amino Acid Sequence; D020029:Base Pairing; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase