A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308.
Repair of DNA interstrand cross-links is a challenging problem for cells. Many human gene products influence sensitivity to DNA cross-linking agents, but the mechanisms of cross-link repair are unknown. In Drosophila melanogaster, the mus308 mutation leads to marked sensitivity to DNA cross-linking agents. The C-terminal portion of the Mus308 polypeptide encodes a DNA polymerase, whereas a putative DNA helicase is encoded by the N-terminal portion. As a step toward isolating proteins involved in DNA cross-link repair, we searched for mammalian genes similar to the DNA helicase portion of Mus308. Human and mouse homologs were isolated from cDNA expression libraries and designated HEL308. Human HEL308 is on chromosome 4q21 and encodes a polypeptide of 1101 amino acids. The protein was expressed in insect cells and purified. HEL308 is a single-stranded DNA-dependent ATPase and DNA helicase. Mutation of a highly conserved lysine to methionine in helicase domain I eliminated both activities. The protein readily displaces 20- to 40-mer duplex oligonucleotides. Displacement of longer substrates was less efficient but was stimulated by the single-stranded DNA-binding protein RPA. Activity was supported by ATP or dATP but not other nucleotide triphosphates. The enzyme translocates on DNA with 3' to 5' polarity and behaves as a multimer upon gel filtration.
Marini
Federica
F
University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15261, USA.
Wood
Richard D
RD
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002894:Chromosomes, Human, Pair 4; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004256:DNA Polymerase I / Q000737:chemistry*; D045643:DNA Repair Enzymes; D018076:DNA, Complementary / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006801:Humans; D007313:Insecta; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D000097462:DNA Polymerase theta
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D003432:Cross-Linking Reagents; D004247:DNA; D004265:DNA Helicases; D004256:DNA Polymerase I; D045643:DNA Repair Enzymes; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; D029721:Drosophila Proteins; D008239:Lysine; D008715:Methionine; C101782:DNApol-theta protein, Drosophila; D000097462:DNA Polymerase theta; C451406:HELQ protein, human