Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Howard Hughes Medical Institute, The Rockefeller University, USA.
Journal of Visualized Experiments (Impact Factor: 1.33). 01/2010; 327(38). DOI: 10.3791/1919
Source: PubMed


In vivo studies suggest that replication forks are arrested by encounters with head-on transcription complexes. Yet, the fate
of the replisome and RNA polymerase (RNAP) after a head-on collision is unknown. We found that the Escherichia coli replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains
highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also found that the transcription-repair
coupling factor Mfd promotes direct restart of the fork after the collision by facilitating displacement of the RNAP. These
findings demonstrate the intrinsic stability of the replication apparatus and a previously unknown role for the transcription-coupled
repair pathway in promoting replication past a RNAP block.

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    • "Whether paused replisomes resume duplication or lose activity is determined by the stability of paused replisomes and the rate of spontaneous or induced dissociation of the blocking nucleoprotein complex. Reconstituted E. coli replisomes retain function for at least a few minutes regardless of the source of the block [41] [42] [43] [44] providing a window of opportunity for removal of the replicative barrier. If this window passes, the pathways by which paused replisomes lose function are unknown but the result is that the replisome must be reassembled back onto the chromosome, a process that is important not only in bacteria but also in eukaryotes [2,45– 47]. "
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    ABSTRACT: Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes. Removal of these barriers demands energy input to not only separate the DNA strands but also to disrupt the multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein-DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability.
    Journal of Molecular Biology 10/2014; 426(24). DOI:10.1016/j.jmb.2014.10.001 · 4.33 Impact Factor
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    • "This pause extends for up to several minutes in the absence of accessory helicase [8]. Other studies indicate that paused replisomes remain stable and eventually resume elongation after displacing RNAP from the DNA template [28, 29]. "
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    ABSTRACT: Replication and transcription are key aspects of DNA metabolism that take place on the same template and potentially interfere with each other. Conflicts between these two activities include head-on or co-directional collisions between DNA and RNA polymerases, which can lead to the formation of DNA breaks and chromosome rearrangements. To avoid these deleterious consequences and prevent genomic instability, cells have evolved multiple mechanisms preventing replication forks from colliding with the transcription machinery. Yet, recent reports indicate that interference between replication and transcription is not limited to physical interactions between polymerases and that other cotranscriptional processes can interfere with DNA replication. These include DNA-RNA hybrids that assemble behind elongating RNA polymerases, impede fork progression and promote homologous recombination. Here, we discuss recent evidence indicating that R-loops represent a major source of genomic instability in all organisms, from bacteria to human, and are potentially implicated in cancer development.
    Current Genomics 03/2012; 13(1):65-73. DOI:10.2174/138920212799034767 · 2.34 Impact Factor
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    • "The inability of Mfd in vivo to release Nun-arrested ECs ahead of replisomes approaching head-on contrasts with a recent report indicating that head-on collisions between replication and transcription can be resolved by Mfd in vitro (Pomerantz and O'Donnell, 2010). "
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    ABSTRACT: Frequent codirectional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that, in E. coli, the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Codirectional collisions with backtracked (arrested) ECs lead to DNA double-strand breaks (DSBs), whereas head-on collisions do not. A mechanistic model is proposed to explain backtracking-mediated DSBs. We further show that bacteria employ various strategies to avoid replisome collisions with backtracked RNAP, the most general of which is translation that prevents RNAP backtracking. If translation is abrogated, DSBs are suppressed by elongation factors that either prevent backtracking or reactivate backtracked ECs. Finally, termination factors also contribute to genomic stability by removing arrested ECs. Our results establish RNAP backtracking as the intrinsic hazard to chromosomal integrity and implicate active ribosomes and other anti-backtracking mechanisms in genome maintenance.
    Cell 08/2011; 146(4):533-43. DOI:10.1016/j.cell.2011.07.034 · 32.24 Impact Factor
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