Cargando…

Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics

Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chai...

Descripción completa

Detalles Bibliográficos
Autores principales: Xia, Shuangluo, Wood, Marcus, Bradley, Michael J., De La Cruz, Enrique M., Konigsberg, William H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799440/
https://www.ncbi.nlm.nih.gov/pubmed/23921641
http://dx.doi.org/10.1093/nar/gkt674
_version_ 1782287868427763712
author Xia, Shuangluo
Wood, Marcus
Bradley, Michael J.
De La Cruz, Enrique M.
Konigsberg, William H.
author_facet Xia, Shuangluo
Wood, Marcus
Bradley, Michael J.
De La Cruz, Enrique M.
Konigsberg, William H.
author_sort Xia, Shuangluo
collection PubMed
description Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chain. Replacement of this residue with Gly or Met in other B family pols resulted in higher mutation rates. When similar substitutions for L415 were introduced into RB69pol, only L415A and L415G had dramatic effects on pre-steady-state kinetic parameters, reducing base selectivity by several hundred fold. On the other hand, the L415M variant behaved like the wild-type. Using a novel tC(o)-tC(nitro) Förster Resonance Energy Transfer (FRET) assay, we were able to show that the partition of the primer terminus between pol and exonuclease (exo) domains was compromised with the L415A and L415G mutants, but not with the L415M variant. These results could be rationalized by changes in their structures as determined by high resolution X-ray crystallography.
format Online
Article
Text
id pubmed-3799440
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-37994402013-10-21 Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics Xia, Shuangluo Wood, Marcus Bradley, Michael J. De La Cruz, Enrique M. Konigsberg, William H. Nucleic Acids Res Nucleic Acid Enzymes Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chain. Replacement of this residue with Gly or Met in other B family pols resulted in higher mutation rates. When similar substitutions for L415 were introduced into RB69pol, only L415A and L415G had dramatic effects on pre-steady-state kinetic parameters, reducing base selectivity by several hundred fold. On the other hand, the L415M variant behaved like the wild-type. Using a novel tC(o)-tC(nitro) Förster Resonance Energy Transfer (FRET) assay, we were able to show that the partition of the primer terminus between pol and exonuclease (exo) domains was compromised with the L415A and L415G mutants, but not with the L415M variant. These results could be rationalized by changes in their structures as determined by high resolution X-ray crystallography. Oxford University Press 2013-10 2013-08-05 /pmc/articles/PMC3799440/ /pubmed/23921641 http://dx.doi.org/10.1093/nar/gkt674 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Nucleic Acid Enzymes
Xia, Shuangluo
Wood, Marcus
Bradley, Michael J.
De La Cruz, Enrique M.
Konigsberg, William H.
Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
title Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
title_full Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
title_fullStr Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
title_full_unstemmed Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
title_short Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics
title_sort alteration in the cavity size adjacent to the active site of rb69 dna polymerase changes its conformational dynamics
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799440/
https://www.ncbi.nlm.nih.gov/pubmed/23921641
http://dx.doi.org/10.1093/nar/gkt674
work_keys_str_mv AT xiashuangluo alterationinthecavitysizeadjacenttotheactivesiteofrb69dnapolymerasechangesitsconformationaldynamics
AT woodmarcus alterationinthecavitysizeadjacenttotheactivesiteofrb69dnapolymerasechangesitsconformationaldynamics
AT bradleymichaelj alterationinthecavitysizeadjacenttotheactivesiteofrb69dnapolymerasechangesitsconformationaldynamics
AT delacruzenriquem alterationinthecavitysizeadjacenttotheactivesiteofrb69dnapolymerasechangesitsconformationaldynamics
AT konigsbergwilliamh alterationinthecavitysizeadjacenttotheactivesiteofrb69dnapolymerasechangesitsconformationaldynamics