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. 2017 Feb 13;45(8):4550–4563. doi: 10.1093/nar/gkx096

Figure 4.

Figure 4.

Analyses of proteins directly bound to the 3΄ primer end at a primer–template junction during PCNA loading with CTF18-RFC–p261N complex by photo-crosslinking. (A) Substrate DNA “APB-Junction” for photo-crosslinking analyses has azidophenacyl bromide (APB) at the 3΄ primer end of the primer–template junction. Two 32P-TMP, and one S-dCMP, were incorporated at the 3΄ primer end of RF30 annealed to TEMP90-R, and a photoreactive crosslinker APB was conjugated to S-dCMP. The control DNA “APB-End” has APB at the blunted end. (B) Photo-crosslinking of 25 fmol APB-Junction (Junction; lanes 1–13) or APB-End (End; lanes 14–16) at 60 mM NaCl by 150 fmol of RFC (lanes 2–16) and 500 fmol of PCNA (lanes 11–16). A set of results without nucleotide (−) or with 2 mM ATP or 250 μM ATPγS (γS) is shown for each condition. Samples with (lanes 1–4, 8–16) or without (lanes 5–7) UV irradiation and with (lanes 8–16) or without (lanes 1–7) nuclease treatment were separated by SDS-PAGE and visualised. (C) Photo-crosslinking of 25 fmol of APB-Junction at 60 mM (lanes 1–3) or 10 mM (lanes 4–9) NaCl by 150 fmol of CTF18-RFC with or without 500 fmol of PCNA, as indicated. A set of results without nucleotides (−) or with 2 mM ATP or 250 μM ATPγS is shown for each condition. (D) Photo-crosslinking of 25 fmol of APB-Junction with combinations of 150 fmol of CTF18-RFC, 250 fmol of CTF18-RFC(5) and 150 fmol of p261Nexo–, as indicated, in the presence of 500 fmol of PCNA and 250 μM ATPγS. (E) Crosslinked bands in (D) corresponding to p261Nexo– and CTF18 were quantified, and their relative intensities were graphed on the right using the highest intensity bands (lane 4) as reference (1.0), with mean ± S.E. of three experimental replicates. “p261N”, “CTF18(7)” and “CTF18(5)” represent p261Nexo–, CTF18-RFC and CTF18-RFC(5), respectively. (F) Photo-crosslinking of the 25 fmol APB-Junction, with 150 fmol of p261Nexo–, 150 fmol of CTF18-RFC and 500 fmol of PCNA in the presence or absence of 2 mM ATP or 250 μM ATPγS. Crosslinked bands corresponding to p261Nexo– and CTF18 were quantified, and the relative values of the CTF18:p261Nexo– ratios are indicated below, with the ratio with ATPγS as 1.0.