Figure 5.

R-loops on the lagging-strand template are not a significant block to replication. (A) Time course of replisome collisions with three different R-loop species on the lagging-strand template (n = 3). (i) Analysis of [α-³²P]dATP-labeled replication reaction products on denaturing agarose gels. There is no DNA replication in the absence of the replication proteins as measured by the incorporation of [α-³²P]dATP into acid-insoluble product. Thus, these samples, lanes 1, 5, and 9, were not included on the denaturing gel that is visualized by autoradiography. Native agarose gel of replication reaction products either stained with (ii) ethidium bromide or (iii) autoradiography of [α-³²P]GTP-labeled RNA in the R-loops. Lanes 1–4, 19mer R-loop (on template HO₁₉); lanes 5–8, 100mer R-loop (HO₁₀₀); lanes 9–12, R-loop array (HO₁₀₀). (B) Both RNase H and RNase A relieve stalling on the HO R-loop array template. Denaturing gel analysis of the replication products of collisions with either the (i) HO 100mer R-loop template or (ii) HO R-loop array template subsequent to treatment with RNase H (lanes 3 and 4), RNase A (lanes 5 and 6), or no enzyme (lanes 1 and 2). (iii) Quantification of the fraction of stall product (n = 3, mean ± standard deviation).