Figure 3
Agnoprotein inhibits in vitro DNA replication mediated by PCNA:DNApolymerase δ. (A) Left panel: In vitro DNA synthesis by PCNA plus pol δ was monitored in the presence of increasing amounts of agnoprotein. DNA synthesis in the presence of pol δ alone was arbitrary set as 1 and the increase by adding PCNA is shown as fold PCNA stimulation. Right panel: Pol λ-mediated DNA synthesis was monitored in the presence of variable nM ratios of agnoprotein:PCNA. Incorporation of radioactivity was measured by scintillation counting. (B) DNA synthesis was assayed by monitoring elongation of a partially double-stranded 39:72 oligonucleotide dimer (=template DNA). Lanes 1 and 16: 32P-labelled template DNA; lanes 2–5: PCNA and Pol δ were allowed to interact before template DNA and increasing amounts of agnoprotein were added; lanes 6–9: agnoprotein and pol δ were pre-incubated before supplementing PCNA and template DNA; lanes 10–14: agnoprotein and PCNA were mixed prior to the addition of pol δ and template DNA; lane 15: PCNA and template DNA; lane 17: template DNA and pol δ. The upper symbol (=) represents the elongated DNA template, while the lower symbol (− ) is the template. (C) Pol λ-mediated DNA synthesis in the presence of PCNA or agnoprotein. The upper band is ssDNA, the middle band is dsDNA, while the lower band represents incomplete dsDNA. Lanes 2–6: increasing amounts of PCNA were added; lanes 9–14: increasing amounts of agnoprotein were added; lane 1: template DNA; lane 8: DNA was incubated with only pol λ. DNA synthesis was visualized by autoradiography as described in 3B. The upper symbol (=) represents the elongated DNA template, while the lower symbol (− ) is the template.