Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase eta from Arabidopsis thaliana.
DNA polymerase eta belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol eta homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol eta activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.
Santiago
María Jesús
MJ
Departamento de Genética, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de Córdoba, Spain.
Alejandre-Durán
Encarna
E
Ruiz-Rubio
Manuel
M
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D017122:Bacteriophage T4 / Q000254:growth & development; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects / Q000821:virology; D016153:Genes, Suppressor / Q000528:radiation effects; D005816:Genetic Complementation Test / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000528:radiation effects*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics; D011740:Pyrimidine Dimers / Q000528:radiation effects; D012351:RNA, Transfer, Gln / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000528:radiation effects; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; D010940:Plant Proteins; D011740:Pyrimidine Dimers; D012351:RNA, Transfer, Gln; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases