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The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site
Open AccessFeature PaperArticle

Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Nicolás Cabrera, 1, 28049 Madrid, Spain
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Biomolecules 2020, 10(2), 203; https://doi.org/10.3390/biom10020203
Received: 3 January 2020 / Revised: 24 January 2020 / Accepted: 27 January 2020 / Published: 30 January 2020
The catalytic active site of the Polymerization Domain (PolDom) of bacterial Ligase D is designed to promote realignments of the primer and template strands and extend mispaired 3′ ends. These features, together with the preferred use of ribonucleotides (NTPs) over deoxynucleotides (dNTPs), allow PolDom to perform efficient double strand break repair by nonhomologous end joining when only a copy of the chromosome is present and the intracellular pool of dNTPs is depleted. Here, we evaluate (i) the role of conserved histidine and serine/threonine residues in NTP insertion, and (ii) the importance in the polymerization reaction of a conserved lysine residue that interacts with the templating nucleotide. To that extent, we have analyzed the biochemical properties of variants at the corresponding His651, Ser768, and Lys606 of Pseudomonas aeruginosa PolDom (Pa-PolDom). The results show that preferential insertion of NMPs is principally due to the histidine that also contributes to the plasticity of the active site to misinsert nucleotides. Additionally, Pa-PolDom Lys606 stabilizes primer dislocations. Finally, we show that the active site of PolDom allows the efficient use of 7,8-dihydro-8-oxo-riboguanosine triphosphate (8oxoGTP) as substrate, a major nucleotide lesion that results from oxidative stress, inserting with the same efficiency both the anti and syn conformations of 8oxoGMP. View Full-Text
Keywords: nonhomologous end joining (NHEJ); archaea/eukaryotic primase; DNA repair; ligase D nonhomologous end joining (NHEJ); archaea/eukaryotic primase; DNA repair; ligase D
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Sánchez-Salvador, A.; de Vega, M. Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom. Biomolecules 2020, 10, 203.

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