Figure 1. Production and characterization of SUMO-modified PCNA.

(a) Diagram of the two polypeptides used to make split ^SUMOPCNA. (b) ^SUMOPCNA supports cell viability. The coding regions of full length PCNA or the N-fragment were cloned into pRS315 (CEN LEU2) with a 500 base pair upstream region containing the native PCNA promoter. The coding region of the ^SUMOC-fragment with the proline-glycine linker was cloned into pRS313 (CEN HIS3) under control of the native PCNA promoter. These constructs were transformed into an EMY74.7 yeast strain with a POL30 gene deletion supported by the wild-type POL30 gene in pTB366 (URA3) under control of its native promoter as described²⁸. Cell growth following counterselection with 5′-fluoroorotic acid showed that the ^SUMOPCNA constructs support cell viability. Shown are the results of spotting 10 μl of saturated yeast cultures, 1:10 dilutions, and 1:100 dilutions on selective growth media overnight. (c) ^SUMOPCNA confers high processivity to pol δ. ^SUMOPCNA was expressed in Rosetta-2 (DE3) cells from a pET-DUET-1 vector in which the Flag-tagged N-fragment was inserted into multi-cloning site 1 and the His₆-tagged ^SUMOC fragment with the glycine-glycine linker was inserted into multi-cloning site 2. ^SUMOPCNA was purified using an NTA-agarose affinity chromatography column (Qiagen), anti-Flag M2 affinity chromatography column (Sigma), and a Superose 6 size exclusion chromatography column (Pharmacia GE Healthcare). We obtained ∼2–3 mg of pure protein per L of cells. Polymerase assays were carried out in the absence of PCNA and in the presence of unmodified PCNA (100 nM), or ^SUMOPCNA (100 nM) as described previously³¹. The reactions contained 50 nM of trimeric pol δ (exo⁺), which was purified as described⁴², 25 nM DNA, and 100 μM dNTP and were stopped after 30 min. The asterisk indicates gel bands corresponding to full length products. (d) ^SUMOPCNA increases the ability of pol δ to incorporate nucleotides opposite a template abasic site. Reactions were carried out as described above. The arrow indicates the gel band corresponding to incorporation opposite the abasic site, and the asterisk indicates gel bands corresponding to full length products.