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. 2007 Oct 11;35(20):6904–6916. doi: 10.1093/nar/gkm822

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© 2007 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Dynamics of replication factors in the elongation phase of DNA synthesis with pol δ*. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ* (1.9 μg) and POLD3 (0.5 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Reconstitution of pol δ with POLD3 and pol δ*. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for pol δ* (70 ng) or pol δ (90 ng) in the absence (−) or presence (+) of POLD3 (20 ng). (C–E) Titration of pol δ* (C), RFC (D) and PCNA (E). Amounts of pol δ* were 0 ng (lane 1), 4.3 ng (lane 2), 17 ng (lane 3), 35 ng (lane 4), 70 ng (lane 5), 100 ng (lane 6), and 140 ng (lane 7). Amounts of RFC used in the titration were 0 ng (lane 1), 2.3 ng (lane 2), 4.7 ng, (lane 3), 9.4 ng (lane 4), 19 ng (lane 5), 38 ng (lane 6) and 75 ng (lane 7). Amounts of PCNA used in titration were 0 ng (lane 1), 5.4 ng (lane 2), 11 ng (lane 3), 22 ng (lane 4), 43 ng (lane 5), 86 ng (lane 6) and 129 ng (lane 7). (F) Titration of PCNA in the presence of HincII. Amount of PCNA is same as (E). Reactions in (C) were carried out for 10 min under the conditions described in the Materials and Methods section. Reactions in (D–F) were carried out for 10 min under the conditions described in the Materials and Methods section except for the amount of pol δ* (140 ng). Products were analyzed by 0.7% alkaline-agarose gel electrophoresis and incorporation of dNMP were measured as described.