Research ArticleBIOCHEMISTRY

Structure and mechanism of human PrimPol, a DNA polymerase with primase activity

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Science Advances  21 Oct 2016:
Vol. 2, no. 10, e1601317
DOI: 10.1126/sciadv.1601317
  • Fig. 1 Overall structure of human PrimPol ternary complex with template-primer DNA and incoming dATP.

    The N-helix and modules ModN and ModC are shown in cartoon representation in dark blue, yellow, and cyan, respectively. The DNA is shown as gray sticks, and the Ca2+ ion is shown as a light blue sphere. The templating base T and the incoming dATP are shown in red. Yellow and cyan dashed lines depict unstructured regions in the ModN and ModC, respectively. The side chains of key catalytic active-site residues Asp114, Glu116, and Asp280 are highlighted in red. Secondary structure elements (α helices and β strands) are labeled in black.

  • Fig. 2 PrimPol active-site region.

    (A) Close-up view of the PrimPol active-site region. The N-helix and the modules ModN and ModC are shown in dark blue, yellow, and cyan, respectively. The DNA is colored gray, the templating base and the incoming dATP are in red, and the Ca2+ ion is in light blue. The key catalytic residues (Asp114, Glu116, and Asp280), the residues contacting the incoming dATP (Lys165, Ser167, His169, Arg288, Asn289, Phe290, Arg291, and Lys297), the templating base (Gly74, Gln75, and Arg76), and the rest of the template strand (Lys10, His46, and Arg47) are shown in sticks and with oxygen atoms in bright red and nitrogen atoms in blue. (B) Details of PrimPol interactions with dATP. The 3′-OH of the dATP forms a hydrogen bond with the backbone amino group of Arg291. Modeling of ATP (2′-OH shown in black) indicates an unfavorable clash between 2′-OH and the backbone carboxyl oxygen of Asn289.

  • Fig. 3 PrimPol lacks space in its active-site cleft to accommodate UV-induced DNA lesions.

    (A) Structure of the yeast Polη inserting dATP opposite the 3′ base of the cis-syn thymine dimer in the template strand [Protein Data Bank (PDB) ID: 3MFI] (21). The fingers, palm, and PAD domains of Polη are colored yellow, cyan, and green, respectively; DNA is in gray, dATP is in red, and the cis-syn thymine dimer is in orange. This structure reveals an enlarged active site that accommodates the two covalently linked thymine bases. (B) Model of PrimPol with a CPD T-T dimer. The model is derived upon a superposition of PrimPol and yPolη (PDB ID: 3MFI) by the key active-site residues. The 5′ base of the CPD T-T dimer severely collides with the backbone atoms of Gly74, Gln75, and Arg76 of the PrimPol’s ModN. The N-helix, ModN, and ModC subdomain residues are shown in dark blue, yellow, and cyan, respectively. (C) Model of PrimPol with a (6–4) pyrimidine-pyrimidone T-T dimer. This model has been produced by a superposition of the undamaged portion of the (6–4) T-T–containing DNA duplex (PDB ID: 1CFL) (43) to the DNA in the PrimPol active site. The 5′ base of the (6–4) T-T dimer severely collides with the backbone atoms of Asp73, Gly74, Gln75, and Arg76 of the PrimPol’s ModN.

  • Fig. 4 PrimPol has room for dNTP at the initiation site.

    (A) A dNTP can be modeled at the initiation site (the oxygen atoms are colored bright red and the phosphorus atoms are in orange) without any clashes. This initiating dNTP becomes the 5′ end of the primer. To model the dNTP, we superimposed the base and sugar moieties of dATP on the base and sugar moieties of the 3′-terminal primer base (gray sticks). Thus, a phosphodiester bond between the incoming dNTP at the elongation site and the “initiating” dNTP can be formed to produce a primer strand with a terminal 5′-triphosphate. (B) A triphosphate of the initiating dNTP does not collide with any residues of PrimPol’s ModC.

  • Fig. 5 PrimPol interactions with the template strand.

    The N-helix, ModN, and ModC residues are shown in dark blue, yellow, and cyan, respectively. The DNA is colored gray, and the templating base T3 is in red. The oxygen atoms of the backbone phosphate groups and O4′ of sugars are highlighted in bright red.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1601317/DC1

    fig. S1. Structure-based sequence alignment of the catalytic core domains of human, mouse, gecko, and zebrafish PrimPols.

    fig. S2. Overall structures of PrimPol and yPolη ternary complexes with template-primer DNA and dNTP.

    fig. S3. Simulated annealing Fo-Fc omit map for dATP, templating base T3, and Ca2+ residues within the PrimPol active-site region.

    fig. S4. Overall structures of human PrimPol-DNA-dNTP complex and human primase PriS complex with uridine triphosphate.

    table S1. Crystallographic parameters and refinement statistics.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Structure-based sequence alignment of the catalytic core domains of human, mouse, gecko, and zebrafish PrimPols.
    • fig. S2. Overall structures of PrimPol and yPolη ternary complexes with template-primer DNA and dNTP.
    • fig. S3. Simulated annealing Fo-Fc omit map for dATP, templating base T3, and Ca2+ residues within the PrimPol active-site region.
    • fig. S4. Overall structures of human PrimPol-DNA-dNTP complex and human primase PriS complex with uridine triphosphate.
    • table S1. Crystallographic parameters and refinement statistics.

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