title: Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction.
abstract: DNA polymerases (pols) are sophisticated protein machines operating in the replication, repair and recombination of genetic material in the complex environment of the cell. DNA pol reactions require at least two divalent metal ions for the phosphodiester bond formation. We explore two understudied roles of metals in pol transactions with emphasis on polα, a crucial enzyme in the initiation of DNA synthesis. We present evidence that the combination of many factors, including the structure of the template/primer, the identity of the metal, the metal turnover in the pol active site, and the influence of the concentration of nucleoside triphosphates, affect DNA pol synthesis. On the poly-dT70 template, the increase of Mg(2+) concentration within the range typically used for pol reactions led to the severe loss of the ability of pol to extend DNA primers and led to a decline in DNA product sizes when extending RNA primers, simulating the effect of "counting" of the number of nucleotides in nascent primers by polα. We suggest that a high Mg(2+) concentration promotes the dynamic formation of unconventional DNA structure(s), thus limiting the apparent processivity of the enzyme. Next, we found that Zn(2+) supported robust polα reactions when the concentration of nucleotides was above the concentration of ions; however, there was only one nucleotide incorporation by the Klenow fragment of DNA pol I. Zn(2+) drastically inhibited polα, but had no effect on Klenow, when Mg(2+) was also present. It is possible that Zn(2+) perturbs metal-mediated transactions in pol active site, for example affecting the step of pyrophosphate removal at the end of each pol cycle necessary for continuation of polymerization.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, United States; Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, United States.
authors.forename: Yinbo
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, United States.
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Baranovskiy
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, United States.
authors.forename: Emin T
authors.initials: ET
authors.lastname: Tahirov
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, United States. Electronic address: ttahirov@unmc.edu.
authors.forename: Tahir H
authors.initials: TH
authors.lastname: Tahirov
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, United States; Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, United States; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, United States. Electronic address: ypavlov@unmc.edu.
authors.forename: Youri I
authors.initials: YI
authors.lastname: Pavlov
mesh_terms: D020134:Catalytic Domain; D002413:Cations, Divalent; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D006801:Humans; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D017433:Protein Structure, Secondary; D013698:Templates, Genetic; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D017931:DNA Primers; D009711:Nucleotides; D004247:DNA; D004256:DNA Polymerase I; D008274:Magnesium; D015032:Zinc
keywords: DNA polymerase alpha; DNA polymerases; Homopolymeric runs; Magnesium; Manganese; Non-B DNA; Polymerase active site; Primase; Zinc