The replisome uses mRNA as a primer after colliding with RNA polymerase

Abstract

Replication forks are impeded by DNA damage and protein-nucleic acid complexes such as transcribing RNA polymerase. For example, head-on collision of the replisome with RNA polymerase results in replication fork arrest. However, co-directional collision of the replisome with RNA polymerase has little or no effect on fork progression. Here we examine co-directional collisions between a replisome and RNA polymerase in vitro. We show that the Escherichia coli replisome uses the RNA transcript as a primer to continue leading-strand synthesis after the collision with RNA polymerase that is displaced from the DNA. This action results in a discontinuity in the leading strand, yet the replisome remains intact and bound to DNA during the entire process. These findings underscore the notable plasticity by which the replisome operates to circumvent obstacles in its path and may explain why the leading strand is synthesized discontinuously in vivo.

Figure 1. Leading strand synthesis is interrupted by a co-directional RNA polymerase

a, Schematic of replisome components and a co-directional RNAP. Replisome proteins include: Pol III core (orange), β-clamp (dark blue), DnaB (yellow), and the clamp-loader (light blue). Primase was omitted from reactions and the lagging strand polymerase is not pictured. b, A 2.2 kb template was constructed which supports leading strand synthesis and co-directional transcription. c,d Leading strand synthesis was performed in the presence of increasing concentrations of a RNAP open and halted elongation complex, respectively. Radio-labeled DNA products were analyzed in alkaline agarose gels (c,d).

Figure 2. The replisome extends the transcript of a co-directional RNA polymerase

a, A co-directional collision of the replisome with a halted RNAP was performed. Extension of the RNA was permitted (lane 1) or blocked (lane 2) by the addition of RNA chain terminator 3′dCTP. RNA sequences are indicated. b, The transcript was radio-labeled by the addition of ³²P-α-GTP and ³²P-α-ATP and analyzed by urea-PAGE prior to (lane 1) and following (lane 2) replication. c, A co-directional collision was performed on a template that either includes (lane 1) or lacks (lane 2) a fork structure. Radio-labeled DNA products were analyzed in alkaline agarose gels (a,c).

Figure 3. The replisome remains intact and displaces a co-directional RNA polymerase from the DNA

a, A His-tagged RNAP halted elongation complex was assembled and immobilized to Ni²⁺ beads. Excess RNAP and DNA were removed by washing followed by replication initiation. Supernatant and pellet fractions were analyzed in an alkaline agarose gel (left). A His-tagged RNAP halted elongation complex was assembled and immobilized as in the left panel, however, replication was not initiated. Supernatant and pellet fractions were analyzed in a non-denaturing agarose gel stained with ethidium bromide (right). b, Leading strand synthesis was performed in solid-phase following the removal of excess Pol III* and DnaB (lanes 1 and 2) either in the presence (lanes 2 and 3) or absence (lane 1) of a co-directional halted RNAP. Radio-labeled DNA products were analyzed in an alkaline agarose gel.

Figure 4. Replisome bypass of a co-directional E. coli RNAP elongation complex

Leading strand synthesis was performed in solid-phase on a 3.5 kb template that either includes (lane 2) or lacks (lane 1) a co-directional halted E. coli RNAP elongation complex. Radio-labeled DNA products were analyzed in an alkaline agarose gel. Percentage of full-length product is indicated and was calculated as described in the methods section.

Figure 5. Model of replisome bypass of a co-directional RNA polymerase

a, The replisome encounters a co-directional RNAP. b, RNAP is displaced from the DNA. The lagging strand polymerase dissociates from the β-clamp and DNA while remaining bound to the clamp-loader. DnaB remains bound to the lagging strand. c, The clamp-loader assembles a new β-clamp at the 3′ terminus of the RNA-DNA hybrid. d, The leading strand polymerase binds to the newly assembled β-clamp. e, The leading strand polymerase extends the mRNA leaving behind a nick or gap in the leading strand.