Interplay Between DNA Polymerase, RNA Polymerase, and RNase H1 During Head-On Transcription-Replication Conflict.

Transcription-replication conflicts (TRCs) often occur in cells and cause DNA replication fork stalling. In this study, we investigated the interplay of RNA polymerase (RNAP), DNA polymerase, and RNase H1 (RH1) during head-on TRC in vitro with precise control over the reaction conditions. We show that it is a catalytically competent transcription elongation complex (TEC) that interferes with the action of both the Klenow fragment and full-length DNA Pol I. An incompetent RNAP complex with an R-loop stimulates the 3'→5' exonuclease activity and pauses the DNA polymerase during head-on TRC. As RNAP advances along the DNA template, elongating the RNA, the head-on TRC is slowly overcome in our model system, likely through the reassociation of the displaced DNA polymerase with the nontemplate DNA strand upstream of RNAP. An isolated R-loop containing an 11-nt heteroduplex (R-loop-11) does not interfere with DNA replication by the Klenow fragment. For DNA Pol I, such an R-loop also does not stall replication but stimulates its 3'→5' exonuclease activity. We demonstrate that a stalled Klenow fragment does not interfere with transcription, whereas a Klenow fragment moving along the TRC substrate towards RNAP alters the kinetics of RNAP. Stalled DNA Pol I does not stop RNAP but stimulates its endonuclease activity. We find that RH1 alone does not displace stalled RNAP from a competent TEC containing R-loop-11 and does not resolve the head-on TRC. On the other hand, RH1 displaces RNAP from the incompetent complex with the TRC substrate. This eliminates the stimulation of the 3'→5' exonuclease activity of DNA polymerase during head-on TRC.