Conflict Resolution in the Genome: How Transcription and Replication Make It Work

Abstract

The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes.

Figure 1. Head-on and co-directional transcription-replication conflicts

A) In prokaryotes, a single origin (oriC) is used to initiate replication in opposite directions along the single circular chromosome. B) In eukaryotes, multiple origins initiate DNA synthesis during S-phase along the linear chromosomes. In both prokaryotic and eukaryotic genomes, head-on conflicts occur when a gene is encoded on the lagging strand, whereas co-directional encounters occur when a gene is encoded on the leading strand. C) Schematic representations of the replisome and ternary RNAP complexes converging on the template DNA in head-on or co-directional orientations. Some key eukaryotic replisome components needed for processive DNA synthesis are indicated, including the replicative helicase (MCM2-7, or DnaB in prokaryotes), leading and lagging strand DNA polymerases (Pol ε/δ), DNA primase (Pol α-Primase), single-strand DNA binding protein (RPA) and clamp loader complex (PCNA).

Figure 2. Coordinating transcription and replication

A) Schematic representation of the E. coli and B. subtilis circular chromosomes. Genes encoded on the (+) and (−) strand are shown in red and black, respectively, with a bias of ~55% or 75% of the genes encoded on the leading strand of replication, respectively. oriC, Replication origin; Ter, Termination region. The chromosome maps were generated with SnapGene. B) Replication fork barriers prevent head-on collisions at the highly transcribed rRNA genes. The yeast rDNA locus consists of the RNAP I transcribed 35S rRNA gene as well as an intergenic spacer region that contains an autonomously replicating sequence (ARS). Although replication is initiated bidirectionally, a replication fork barrier (RFB) bound by the fork blocking protein 1 (Fob1) prevents forks from entering the 3’ end of the rRNA gene. C) Spatial separation of replication and transcription sites throughout S phase. The images show nuclei of mouse 3T3 cells in early, mid or late S-phase pulsed simultaneously with digoxigenin-dUTP or BrUTP to mark active sites of DNA replication or transcription, respectively. The images were modified and reprinted from (Wei et al., 1998) with permission. The cartoon illustrates a possible interpretation of such clusters of high transcription (RNAP complexes in red) or replication activity (replisome complexes in green), supporting the view that the functional organization of the nucleus in space and time can reduce interference between the two machineries.

Figure 3. Co-transcriptional mechanisms to suppress TRCs

A) During transcription, the ternary RNAP complex associates with the DNA template in different functional states and conformations that could lead to different types of transcription blocks. B) Transcription roadblocks may be resolved by distinct pathways. The prokaryotic (black) and eukaryotic (blue) proteins involved are shown. DksA in bacteria and the human RECQL5 helicase reduce stalling or pausing events by controlling the transcription elongation rate. The anti-backtracking factors GreA/GreB or TFIIS promote cleavage of the backtracked transcript and create a new 3’OH group in the active site to resume transcription. RNAP complexes stalled at a site of DNA damage can be removed from the DNA template by TC-NER or proteasome-mediated degradation via poly-ubiquitylation. Transcription termination and resolution of R-loops is mediated by the Rat1/XRN2 exonuclease and RNA:DNA helicases including Rho in prokaryotes and Sen1/SETX or AQR in eukaryotes. In addition, R-loops may be recognized and processed by the TC-NER endonucleases XPF/XPG. All of these co-transcriptional mechanisms help to remove the transcriptional blocks and thereby suppress collisions with replication forks. Yellow star represents the lesion in both panels.

Figure 4. Replication-associated mechanisms to prevent and resolve transcription-replication conflicts

A) Accessory helicases including Rep (E. coli), PcrA (B. subtilis), Rrm3, Sen1 (yeast) or SETX (human) can assist the replicative helicase by dislodging transcription complexes ahead of the replication fork. B) The S-phase checkpoint can monitor and respond to replication forks stalled at transcription complexes in eukaryotic cells. The Mec1/ATR kinase may promote fork progression and stability at transcribed genes by phosphorylating the nucleoporin Mlp1, thereby releasing genes from the nuclear pore to reduce topological tension. The checkpoint also controls tRNA gene transcription mediated by the Maf1 repressor to reduce interference with replication. The osmostress-induced protein kinase Hog1 phosphorylates Mrc1, a downstream component of the Mec1/ATR pathway, thereby preventing early origin firing and fork progression to prevent TRCs during osmostress. In addition, Mec1/ATR, in cooperation with INO80 and PAF1C, can trigger the efficient removal of chromatin-bound RNAPII near early firing origins. NPC, Nuclear Pore Complex C) Replication forks stalled at transcription complexes can resume DNA synthesis by different fork restart and DNA repair pathways. (i) A replication fork stalled at a transcription complex can be rescued by firing of an adjacent dormant origin. This back-up replication fork encounters the transcription complex from the opposite direction and may represent a second chance to remove the transcription block and resume DNA synthesis. Alternatively, replication forks stalled at TRCs may be stabilized by ATR, BRCA2, or the FA-pathway (ii). Prolonged stalling of the replication fork may also promote re-annealing of parental strands priming fork reversal. Removal of the transcription block by one or several of the pathways/factors listed can then promote fork restart (iii). If the transcription block persists, this may ultimately lead to fork breakage (iv). Break-induced replication (BIR) and/or homologous recombination (HR)-dependent repair mechanisms may then be used to overcome the obstacle.