Cargando…
Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom
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 Po...
Autores principales: | , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072297/ https://www.ncbi.nlm.nih.gov/pubmed/32019147 http://dx.doi.org/10.3390/biom10020203 |
_version_ | 1783506373807439872 |
---|---|
author | Sánchez-Salvador, Alejandro de Vega, Miguel |
author_facet | Sánchez-Salvador, Alejandro de Vega, Miguel |
author_sort | Sánchez-Salvador, Alejandro |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7072297 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-70722972020-03-19 Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom Sánchez-Salvador, Alejandro de Vega, Miguel Biomolecules Article 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. MDPI 2020-01-30 /pmc/articles/PMC7072297/ /pubmed/32019147 http://dx.doi.org/10.3390/biom10020203 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Sánchez-Salvador, Alejandro de Vega, Miguel Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom |
title | Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom |
title_full | Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom |
title_fullStr | Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom |
title_full_unstemmed | Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom |
title_short | Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom |
title_sort | structural determinants responsible for the preferential insertion of ribonucleotides by bacterial nhej poldom |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072297/ https://www.ncbi.nlm.nih.gov/pubmed/32019147 http://dx.doi.org/10.3390/biom10020203 |
work_keys_str_mv | AT sanchezsalvadoralejandro structuraldeterminantsresponsibleforthepreferentialinsertionofribonucleotidesbybacterialnhejpoldom AT devegamiguel structuraldeterminantsresponsibleforthepreferentialinsertionofribonucleotidesbybacterialnhejpoldom |