Abstract
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.
Highlights
DNA replication is a process that requires the concerted action of numerous proteins and enzymes
A series of different buffers were continuously pumped through the individual inlets in order to create discrete laminar flowing streams of buffer that differ in pH from one another when traveling between the anode and cathode (Fig 1A and 1B)
Using dot blot analysis for determining the location of proliferating cell nuclear antigen (PCNA) within the fractions eluting from the separation, we showed that PCNA consistently eluted in the same fractions, and the resolving power of interval zone electrophoresis (IZE) was unaffected by increasing the amount of protein loaded into the separation chamber over the six fold range we used in this study
Summary
DNA replication is a process that requires the concerted action of numerous proteins and enzymes. Research reported in this publication included work performed in the Translational Biomarker Discovery Core and instrumentation from the Mass Spectrometry and Proteomics Core that was supported by a Cancer Center support grant from the National Cancer Institute of the National Institutes of Health made to the City of Hope under award number P30CA033572. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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