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

<div><p>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.</p></div

Separation of IZE fractions by BN gel electrophoresis.

<p>(A) The gels were stained with Coomassie. (B) Separate gels were transferred to PVDF membrane and probed with PCNA antibody. Fraction numbers are labeled on top of the figure.</p

Previously identified DNA synthesome proteins or protein subunits identified by mass spectrometry from BN gel with a relative electrophoretic mobility of 0.8–1 MDa.

<p>Previously identified DNA synthesome proteins or protein subunits identified by mass spectrometry from BN gel with a relative electrophoretic mobility of 0.8–1 MDa.</p

In vitro SV40 DNA replication activity relative to the position of PCNA.

<p>(A) Circles with the solid line represent the normalized SV40 DNA replication activity labeled on the left axis. Diamonds with the dotted line represent the protein concentration in μg labeled on the right axis. (B) Squares represent dot blot quantification of PCNA levels from IZE fractions run on the same day. The data were normalized between 0–1.</p

Fractionation of HeLa cell proteins using IZE.

<p>(A) Simplified diagram of the FFE separation chamber. (B) pH measured for every other fraction on the 96-well plate after protein separation. (C) Separation of fractions from the IZE by denaturing gel electrophoresis and detection by silver staining. Fraction numbers are labeled on top, and M represents the mol wt marker. P4 represents the protein before the IZE separation. The molecular masses of the markers are 250 kDa, 150 kDa, 100 kDa, 75 kDa, 50 kDa, 37 kDa, 25 kDa, and 20 kDa labeled A to H from top to bottom.</p

Dot blot analysis of IZE fractions.

<p>(A) Dot blot analysis of an IZE separation from the P4 fraction using antibodies that recognize PCNA, Topo I, Pol ɛ subunit 2, RPA subunit 2, Pol δ catalytic subunit, and RFC subunit 4. Recombinant PCNA (rPCNA) was analyzed following IZE separation of PCNA-FLAG expressed and purified protein. Fraction numbers are labeled at the top of the figure. (B) Dot blot quantification of PCNA. The Western blot signal of each fraction for PCNA from the P4 fraction (black circle) and PCNA-FLAG (blue square) was normalized from 0 to 1.</p