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

Article (PDF Available)inJournal of Visualized Experiments 327(38) · January 2010with14 Reads
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.

Full-text (PDF)

Available from: PubMed Central · License: CC BY
    • "Two types of collisions are possible; head to head or co-directional. Head to head collisions appear to be more detrimental to cell viability686970 since ribosomal operons and other highly transcribed genes are encoded on the leading DNA strand, and therefore transcribed codirectionally with replication fork progression [71]. However, codirectional collisions are still inevitable because the DNA polymerase moves at least 10 times faster than RNAP [72]. "
    [Show abstract] [Hide abstract] ABSTRACT: Transcription elongation is regulated at several different levels, including control by various accessory transcription elongation factors. A distinct group of these factors interacts with the RNA polymerase secondary channel, an opening at the enzyme surface that leads to its active center. Despite investigation for several years, the activities and in vivo roles of some of these factors remain obscure. Here, we review the recent progress in understanding the functions of the secondary channel binding factors in bacteria. In particular, we highlight the surprising role of global regulator DksA in fidelity of RNA synthesis and the resolution of RNA polymerase traffic jams by the Gre factor. These findings indicate a potential link between transcription fidelity and collisions of the transcription and replication machineries.
    Full-text · Article · Jun 2015
    • "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]. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Oct 2014
    • "Extensive backtracking produces arrested ECs that require internal cleavage of the nascent RNA to continue transcription (Nudler et al., 1997; Komissarova and Kashlev, 1997; Shaevitz et al., 2003) and may form barriers to DNA replication that decrease cell viability. The transcription cleavage factors GreA/GreB, which reactivate arrested ECs (Toulmé et al., 2000; Marr and Roberts, 2000), and Rho, which terminates both intragenic and stable RNA transcription (Peters et al., 2009Peters et al., , 2012 ), help relieve replication-transcription conflicts in vivo (Trautinger et al., 2005; Tehranchi et al., 2010; Washburn and Gottesman, 2011; Dutta et al., 2011) and in vitro (Pomerantz and O'Donnell, 2010). In addition to GreA/GreB and Rho, the transcription factor DksA has been implicated in preventing transcription-replication conflicts. "
    [Show abstract] [Hide abstract] ABSTRACT: In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.
    Full-text · Article · Mar 2014
Show more