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

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.

Replication Protein A Enhances Kinetics of Uracil DNA Glycosylase on ssDNA and Across DNA Junctions: Explored with a DNA Repair Complex Produced with SpyCatcher/SpyTag Ligation

“…This led to the finding that proteins organize into distinct replication and repair complexes to enhance the efficiency of their processes. [1][2][3][4][5] One well-examined hub protein is replication protein A (RPA), which binds ssDNA with high affinity and has dozens of protein binding partners. [6,7] RPA recruits other proteins to specific nuclear sites including replication forks, [8][9][10][11][12][13][14][15] and RPA can affect the activity of enzymes and other DNA binding proteins.…”

“…DNA repair proteins including RPA interact with sufficient strength to remain intact as isolated complexes under various chromatography conditions. [1,3] On the other hand, the interactions of recombinant proteins with RPA in vitro typically occur with K d values in the high nM to low μM range. [17,[19][20][21][22] These moderate affinities suggest that RPA-containing protein complexes may be dynamic in composition, at least in cell-free systems.…”

“…Proteins in similar DNA repair pathways often interact with each other, or else interact with a common scaffold or hub protein that binds multiple other proteins. This led to the finding that proteins organize into distinct replication and repair complexes to enhance the efficiency of their processes [1–5] . One well‐examined hub protein is replication protein A (RPA), which binds ssDNA with high affinity and has dozens of protein binding partners [6,7] .…”

Preparative continuous flow electrophoretic instrumentation for purification of biological samples

“…Continuous flow separations that were based on various modes of electrophoresis have been well described in the literature in classical formats [1–6] as well as in miniaturized formats [7–9]. They have been used as a preparative and analytical methods for the separation of various biological samples [9–15]. In continuous flow electrophoresis, the sample is continuously introduced into liquid flow in the separation space and the voltage is applied perpendicularly to the flow to separate the sample components.…”

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

“…An electric field is applied perpendicularly to the sample flow to deflect analytes, laterally according to their mobility or p I s, as they continuously flow through the separation channel . Because of its advantages of mild separation environment and solid support medium free characteristics, FFE has been successfully applied to the separation of various biological samples, such as nucleic acids , peptide , proteins , cell organelles , and so on. Moreover, most of the separation modes of CE can be applied to FFE as well , for example, free‐flow zone electrophoresis (FFZE) , free‐flow isoelectric focusing (FFIEF) , and free‐flow isotachophoresis .…”