title: The 50-kDa primase subunit of Drosophila melanogaster DNA polymerase alpha. Molecular characterization of the gene and functional analysis of the overexpressed protein.
abstract: The gene encoding the 50-kDa subunit of Drosophila melanogaster DNA polymerase alpha has been cloned. A comparison of the predicted polypeptide sequence of the Drosophila protein with the equivalent subunits from mouse and yeast suggests that they are closely related and defines three conserved regions which are likely to be important for enzyme activity. The expression patterns of both the 50-kDa protein and its transcript (a single RNA message of 1.6 kilobases) throughout development are consistent with a role of the protein in DNA replication. When overexpressed and purified the 50-kDa subunit displays DNA primase activity. The products of the reaction, mainly oligoribonucleotides 12-14 nucleotides in length, plus dimers and some trimers, are similar to those synthesized by either the intact DNA polymerase alpha, or the biochemically isolated primase heterodimer. The isolated primase also shows similar sensitivity to antibodies, magnesium and monovalent cations, and the same nucleotide requirements as complexed forms of the primase. The isolated subunit, however, is more thermally labile, suggesting a role for the additional subunits in DNA polymerase alpha in stabilizing the primase activity of the 50-kDa primase subunit.
authors.affiliation: Marie Curie Research Institute, Oxted, Surrey, United Kingdom.
authors.forename: C J
authors.initials: CJ
authors.lastname: Bakkenist
authors.forename: S
authors.initials: S
authors.lastname: Cotterill
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003570:Cytidine Triphosphate / Q000378:metabolism / Q000528:radiation effects; D004257:DNA Polymerase II / Q000235:genetics*; D019915:DNA Primase; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D017344:Genes, Insect / Q000235:genetics*; D015698:Genomic Library; D006358:Hot Temperature; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000096:biosynthesis; D014176:Protein Biosynthesis; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000528:radiation effects; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
chemical_list: D018076:DNA, Complementary; D009843:Oligoribonucleotides; D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II