Figure 3.

Oligomerization state of PCNA at various monomer concentrations and buffer conditions. (a) Gel filtration chromatography for estimation of the molecular sizes of the M. acetivorans PCNA at different concentrations by SMART system (GE Healthcare). The elution volume of the major peak at a concentration of 10 μM trimer was estimated as 83.5 kDa, and as the estimated molecular mass of a monomer of MacPCNA, based on the polypeptide sequence, is 26.6 kDa, the elution volume suggested a protein existing as trimers (83.5:26.6 = 3.1) in solution. The elution volume of the peak at a trimer concentration of 1.7 μM was estimated as representing a protein of molecular mass 74.1 kDa, hence this represented 2.7 monomers of PCNA, which one will assume represents a trimer, and finally the elution volume of the protein injected at 170 nM trimer concentration suggested a molecular mass of 28.5 kDa, which we assumed is a monomer. (b) RFC-induced assembly in our standard reaction buffer (black) and in the gel filtration high salt buffer C plus 10 mM MgCl₂ (gray). Total PCNA concentration is 40 nM. RFC concentration is 40 nM. ATP concentration is 0.5 mM.