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Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA rep...

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Autores principales: Li, Caroline M., Miao, Yunan, Lingeman, Robert G., Hickey, Robert J., Malkas, Linda H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201288/
https://www.ncbi.nlm.nih.gov/pubmed/28036377
http://dx.doi.org/10.1371/journal.pone.0169259
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author Li, Caroline M.
Miao, Yunan
Lingeman, Robert G.
Hickey, Robert J.
Malkas, Linda H.
author_facet Li, Caroline M.
Miao, Yunan
Lingeman, Robert G.
Hickey, Robert J.
Malkas, Linda H.
author_sort Li, Caroline M.
collection PubMed
description We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.
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spelling pubmed-52012882017-01-19 Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis Li, Caroline M. Miao, Yunan Lingeman, Robert G. Hickey, Robert J. Malkas, Linda H. PLoS One Research Article We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits. Public Library of Science 2016-12-30 /pmc/articles/PMC5201288/ /pubmed/28036377 http://dx.doi.org/10.1371/journal.pone.0169259 Text en © 2016 Li et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Li, Caroline M.
Miao, Yunan
Lingeman, Robert G.
Hickey, Robert J.
Malkas, Linda H.
Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis
title Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis
title_full Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis
title_fullStr Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis
title_full_unstemmed Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis
title_short Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis
title_sort partial purification of a megadalton dna replication complex by free flow electrophoresis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201288/
https://www.ncbi.nlm.nih.gov/pubmed/28036377
http://dx.doi.org/10.1371/journal.pone.0169259
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