Figure 3.

Generation of R-loop templates. (A) Extension of the RNA in R-loops by either DnaG or Pol III* is blocked when the 3′-end of the RNA lacks a 3′-OH group. R-loops formed with [α-³²P]GTP-labeled RNA as described in Materials and Methods on the CO₁₉ template either without (G19) or with (3′dC20) incorporation of 3′-dCTP were incubated in replication buffer lacking either the complete complement of replication proteins (all) or either DNA Polymerase III* (III*) or DnaA (A). The products were analyzed by electrophoresis through a 20% 7M urea polyacrylamide gel (n = 2). (B) Schematic of the steps in preparing the R-loop templates as described in Results and Materials and Methods. (C) RNA transcripts present in the 19mer (lane 1), 100mer (lane 2), and R-loop array (lane 3) CO templates as analyzed by denaturing polyacrylamide gel electrophoresis. (D) RNase H, but not RNase A, digests RNA in the R loops. (i) 100mer R-loops and (ii) R-loop arrays formed on template CO₁₀₀ were treated in replication buffer either without (–) or with the indicated RNases (RNase H, 0.1 U/μl; RNase A, 20 μM) for 10 min (n = 3). Native agarose gels of replication reaction products stained with ethidium bromide (DNA) or imaged by autoradiography of [α-³²P]GTP-labeled RNA (mRNA) in the R-loops.