title: Insight into the Human DNA Primase Interaction with Template-Primer.
abstract: DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C. The specific mode of interaction with a template-primer involving the terminal 5'-triphosphate of RNA and the 3'-overhang of DNA results in a stable complex between p58C and the DNA/RNA duplex. Our results explain how p58C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center.
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Baranovskiy
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, Department of Biochemistry and Molecular Biology, and.
authors.forename: Yinbo
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Suwa
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center.
authors.forename: Jianyou
authors.initials: J
authors.lastname: Gu
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center.
authors.forename: Nigar D
authors.initials: ND
authors.lastname: Babayeva
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, Department of Biochemistry and Molecular Biology, and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198.
authors.forename: Youri I
authors.initials: YI
authors.lastname: Pavlov
authors.affiliation: From the Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, ttahirov@unmc.edu.
authors.forename: Tahir H
authors.initials: TH
authors.lastname: Tahirov
mesh_terms: D001665:Binding Sites; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D064251:Multifunctional Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D064251:Multifunctional Enzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012313:RNA; D019915:DNA Primase; C000609421:PRIM1 protein, human; C525356:PRIM2 protein, human; C585901:PrimPol protein, human; D004259:DNA-Directed DNA Polymerase
keywords: 5′-triphosphate; DNA primase; DNA replication; DNA-protein interaction; RNA primer length counting; RNA synthesis; RNA/DNA duplex; gel electrophoresis; human; p58 subunit; primase activity