Cargando…
Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding
BACKGROUND: The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six...
Autores principales: | , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2010
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933578/ https://www.ncbi.nlm.nih.gov/pubmed/20716382 http://dx.doi.org/10.1186/1471-2199-11-62 |
_version_ | 1782186150679543808 |
---|---|
author | Brewster, Aaron S Slaymaker, Ian M Afif, Samir A Chen, Xiaojiang S |
author_facet | Brewster, Aaron S Slaymaker, Ian M Afif, Samir A Chen, Xiaojiang S |
author_sort | Brewster, Aaron S |
collection | PubMed |
description | BACKGROUND: The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. RESULTS: In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. CONCLUSIONS: These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding. |
format | Text |
id | pubmed-2933578 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-29335782010-09-07 Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding Brewster, Aaron S Slaymaker, Ian M Afif, Samir A Chen, Xiaojiang S BMC Mol Biol Research Article BACKGROUND: The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. RESULTS: In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. CONCLUSIONS: These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding. BioMed Central 2010-08-18 /pmc/articles/PMC2933578/ /pubmed/20716382 http://dx.doi.org/10.1186/1471-2199-11-62 Text en Copyright ©2010 Brewster et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Brewster, Aaron S Slaymaker, Ian M Afif, Samir A Chen, Xiaojiang S Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding |
title | Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding |
title_full | Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding |
title_fullStr | Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding |
title_full_unstemmed | Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding |
title_short | Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding |
title_sort | mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and dna binding |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933578/ https://www.ncbi.nlm.nih.gov/pubmed/20716382 http://dx.doi.org/10.1186/1471-2199-11-62 |
work_keys_str_mv | AT brewsteraarons mutationalanalysisofanarchaealminichromosomemaintenanceproteinexteriorhairpinrevealscriticalresiduesforhelicaseactivityanddnabinding AT slaymakerianm mutationalanalysisofanarchaealminichromosomemaintenanceproteinexteriorhairpinrevealscriticalresiduesforhelicaseactivityanddnabinding AT afifsamira mutationalanalysisofanarchaealminichromosomemaintenanceproteinexteriorhairpinrevealscriticalresiduesforhelicaseactivityanddnabinding AT chenxiaojiangs mutationalanalysisofanarchaealminichromosomemaintenanceproteinexteriorhairpinrevealscriticalresiduesforhelicaseactivityanddnabinding |