When DNA Topology Turns Deadly - RNA Polymerases Dig in Their R-Loops to Stand Their Ground: New Positive and Negative (Super)Twists in the Replication-Transcription Conflict

Abstract

Head-on replication-transcription conflict is especially bitter in bacterial chromosomes, explaining why actively transcribed genes are always co-oriented with replication. The mechanism of this conflict remains unclear, besides the anticipated accumulation of positive supercoils between head-on-conflicting polymerases. Unexpectedly, experiments in bacterial and human cells reveal that head-on replication-transcription conflict induces R-loops, indicating hypernegative supercoiling [(-)sc] in the region - precisely the opposite of that assumed. Further, as a result of these R-loops, both replication and transcription in the affected region permanently stall, so the failure of R-loop removal in RNase H-deficient bacteria becomes lethal. How hyper(-)sc emerges in the middle of a positively supercoiled chromosomal domain is a mystery that requires rethinking of topoisomerase action around polymerases.

Keywords: R-loops; RNase H; replication–transcription conflicts; supercoiling; topoisomerases.

Fig. 1. The twin-domain topological nature of DNA replication and transcription

Duplex DNA is shown by double blue lines, RNA is shown as orange lines. The 3′-ends are identified by arrowheads. Position of relaxation of negative and positive supercoils are shown by colored arrows. A. An independent topological domain with a replication factory (green hexagon) that pulls template DNA through (from right to left here), generating positive supercoiling in the template DNA, while releasing negatively supercoiled daughter duplexes. B. An independent topological domain with a transcription factory (green circle), that pulls the template DNA through (from left to right here), generating positive supercoiling ahead of itself, while negative supercoiling behind. C. An independent topological domain with both a replication fork and a head-on transcription unit. Due to the possible distal nature of processive (+)sc relaxation, accumulation of hyper(+)sc between the two factories is suspected.

Fig. 2. The two types of R-loops

A. TEC-free R-loop. B. R-loop-aTEC.

Fig. 3. How replication-transcription conflict could spawn R-loops behind RNA polymerases

The replisome is shown as a hexagon surrounding the replication fork or a Holiday junction. The TEC is shown as a circle surrounding the transcription loop. Blue lines, DNA strands; orange lines, RNA strands. Big blue circles, ribosomes; chains of small circles, nascent polypeptide chains. A. Converging replication and transcription factories find themselves in the head-on conflict. B. Accumulation of positive supercoils ((+)sc) between the two factories stalls both. C. An unspecified event suddenly drops the level of (+)sc in the region, which allows the transcription factory to resume pulling in the template DNA and releasing it with hyper(−)sc behind. D. While the hyper(−)sc behind the TEC, in combination with a slow ribosome restart, invites formation of an R-loop-aTEC, the replication fork remains stalled at the original position due to accumulated (+sc).

Fig. 4. Possible scenarios of the sudden drop in (+)sc between two converging factories

A. and C. These two panels corresponds to Fig. 3B and D, with ribosomes omitted for clarity. B-1. Replication factory loses the contact with the fork. The fork freely rotates as a result, redistributing the (+)sc in the unreplicated DNA as precatenanes behind the fork. B-2. Replication factory malfunctions and allows the replication fork to regress, still holding on to the resulting Holliday junction. B-3. Massive (+)sc attracts distributive topo II enzymes, which remove the bulk of (+)sc, but dissociate due to their low affinity to random DNA, leaving behind enough (+)sc to inhibit the replisome, but not enough to inhibit the TEC.