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RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases.

ABSTRACT: We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this Luria-Bertani (LB)-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoB(H447R) and rpoB(D444G) prevent activation of the Prrn core promoter in rich medium, but only rpoB(H447R) also suppresses the auxotrophy of a relA spoT mutant (stringent-like phenotype). rpoC(H113R) suppresses the thermo-sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoC(P451L) prevent R-loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication-transcription collisions. In a RecF(+) context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG.

SUBMITTER: Baharoglu Z

PROVIDER: S-EPMC2936116 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases.

Baharoglu Zeynep Z Lestini Roxane R Duigou Stéphane S Michel Bénédicte B

Molecular microbiology 20100519 2

We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this Luria-Bertani (LB)-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoB(H447R) and rpoB(D444G) prevent activation of the Prrn core promoter in rich medium ...[more]

PMID: 20497334

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Project description:Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.

Project description:Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

Dataset Information

RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases.

Publications

RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases.

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