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DNA flap creation by the RarA/MgsA protein of Escherichia coli.

ABSTRACT: We identify a novel activity of the RarA (also MgsA) protein of Escherichia coli, demonstrating that this protein functions at DNA ends to generate flaps. A AAA+ ATPase in the clamp loader clade, RarA protein is part of a highly conserved family of DNA metabolism proteins. We demonstrate that RarA binds to double-stranded DNA in its ATP-bound state and single-stranded DNA in its apo state. RarA ATPase activity is stimulated by single-stranded DNA gaps and double-stranded DNA ends. At these double-stranded DNA ends, RarA couples the energy of ATP binding and hydrolysis to separating the strands of duplex DNA, creating flaps. We hypothesize that the creation of a flap at the site of a leading strand discontinuity could, in principle, allow DnaB and the associated replisome to continue DNA synthesis without impediment, with leading strand re-priming by DnaG. Replication forks could thus be rescued in a manner that does not involve replisome disassembly or reassembly, albeit with loss of one of the two chromosomal products of a replication cycle.

SUBMITTER: Stanage TH

PROVIDER: S-EPMC5389604 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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DNA flap creation by the RarA/MgsA protein of Escherichia coli.

Stanage Tyler H TH Page Asher N AN Cox Michael M MM

Nucleic acids research 20170301 5

We identify a novel activity of the RarA (also MgsA) protein of Escherichia coli, demonstrating that this protein functions at DNA ends to generate flaps. A AAA+ ATPase in the clamp loader clade, RarA protein is part of a highly conserved family of DNA metabolism proteins. We demonstrate that RarA binds to double-stranded DNA in its ATP-bound state and single-stranded DNA in its apo state. RarA ATPase activity is stimulated by single-stranded DNA gaps and double-stranded DNA ends. At these doubl ...[more]

PMID: 28053120

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Project description:BackgroundMost bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained by ptsG, mgsA mutants etc., but its mechanism has not been well understood. It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation.ResultsIn order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 h?¹ using wild type Escherichia coli. The result indicates that the transcript levels of global regulators such as crp, cra, mlc and rpoS decreased, while those of fadR, iclR, soxR/S increased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO? evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the ¹³C-flux analysis. In order to make clear the catabolite regulation, the effect of crp gene knockout (?crp) and crp enhancement (crp?) as well as mlc, mgsA, pgi and ptsG gene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h?¹ and by some batch cultures. In the case of ?crp (and also ?mlc) mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case of crp? mutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression of ptsG and ptsH by the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained by mgsA, pgi, and ptsG mutants due to increase in crp as well as cyaA, while glucose consumption rate became lower.ConclusionsThe transcriptional catabolite regulation mechanism was made clear for the wild type E. coli, and its crp, mlc, ptsG, pgi, and mgsA gene knockout mutants. The results indicate that catabolite repression can be relaxed and crp as well as cyaA can be increased by crp?, mgsA, pgi, and ptsG mutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation of ptsG in all the mutants considered.

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DNA flap creation by the RarA/MgsA protein of Escherichia coli.

Publications

DNA flap creation by the RarA/MgsA protein of Escherichia coli.

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