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. 2009 Aug 20;28(19):2981–2991. doi: 10.1038/emboj.2009.233

Figure 1.

Figure 1

Pol III HE does not recognize a 5′ terminus for collision release. (A) Pol III* and β were assembled onto a 7.2-kb primed M13mp18 ssDNA in the presence (or absence) of EBNA1, which binds to the extreme 5′ nucleotides of the 46-mer primed site. In a separate reaction, β is assembled onto a five-fold excess of a challenge primed M13Gori ssDNA (8.6 kb). Reactions were mixed, replication was initiated and timed aliquots were analysed in a native agarose gel. In control reactions, lanes 1–4, β was not assembled onto acceptor M13Gori DNA. Lanes 5–8 are reactions in the absence of EBNA1, and lanes 9–12 contained EBNA1. The 7.2- and 8.6-kb products were quantified using a phosphorimager and are shown in the plot as the ratio of replicated 8.6 kb acceptor RFII DNA over replicated 7.2 kb RFII donor DNA versus time. (B) The 5′ terminus of the primer is modified with either biotin (plus or minus streptavidin), or psoralen (see scheme). Reactions were performed and the 7.2- and 8.6-kb RFII products were quantified as described in (A).