RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

Tashjian, Tommy F.; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M.; Godoy, Veronica G.

doi:10.3389/fmicb.2017.00288

Cited by 8 publications

(5 citation statements)

References 38 publications

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“…We used 90- or 180-bp dsDNAs and ssDNAs of varying lengths of homology (from the heterologous N = 0 up to N = 98) and a total length of 98 nt. For polymerase, we used either 1 μ m Escherichia coli DNA Pol IV (obtained using Pol IV overproducer plasmids (31, 32)) or 5 units of DNA LF- Bsu Pol (New England Biolabs; 5000 units/ml). DNA Pol IV reactions were done in RecA buffer containing 0.1 mg/ml BSA, 2 m m dATP, and 0.4 m m dNTPs, whereas DNA LF- Bsu Pol experiments were performed in RecA buffer containing 1 m m ATP and 30 m m NaCl.…”

Section: Methodsmentioning

confidence: 99%

Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology

Danilowicz

Tashjian

et al. 2019

Journal of Biological Chemistry

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DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks. However, the process of recombinational repair between short DNA regions of accidental similarity can lead to fatal genomic rearrangements. Previous studies have probed how effectively RecA discriminates against interactions involving a short similar sequence that is embedded in otherwise dissimilar sequences but have not yielded fully conclusive results. Here, we present results of in vitro experiments with fluorescent probes strategically located on the interacting DNA fragments used for recombination. Our findings suggest that DNA synthesis increases the stability of the recombination products. Fluorescence measurements can also probe the homology dependence of the extension of invading DNA strands in D-loops formed by RecA-mediated strand exchange. We examined the slow extension of the invading strand in a D-loop by DNA polymerase (Pol) IV and the more rapid extension by DNA polymerase LF- Bsu . We found that when DNA Pol IV extends the invading strand in a D-loop formed by RecA-mediated strand exchange, the extension afforded by 82 bp of homology is significantly longer than the extension on 50 bp of homology. In contrast, the extension of the invading strand in D-loops by DNA LF- Bsu Pol is similar for intermediates with ≥50 bp of homology. These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase.

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Section: Methodsmentioning

confidence: 99%

Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology

Danilowicz

Tashjian

et al. 2019

Journal of Biological Chemistry

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“…Escherichia coli Pol IV was purified from the TMCΔT strain (BL21-AI ΔdinB, ΔumuDC, ΔrecA) by ion exchange chromatography and hydrophobic interaction chromatography as previously described (30,31). Oligonucleotides were obtained from Integrated DNA Technologies (IDT) and are listed in the Supplementary Materials and Methods section.…”

Section: Methodsmentioning

confidence: 99%

The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements

Danilowicz

Tashjian

et al. 2018

Nucleic Acids Research

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Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3′ single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3′ ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA–RecA filaments, each ssDNA–RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3′ end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.

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“…In normally growing cells, Pol IV was shown not to contribute to the error rate of chromosomal DNA replication (Kuban et al, ; Wolff et al, ), most likely due to limited access to the normal replication fork. Tashjian et al () have shown in vitro that DinB performs poor DNA synthesis with RNA primers and that it is additionally impeded upon interaction with RecA. They postulate that poor synthesis of DinB using RNA primers might represent a mechanism to prevent DNA synthesis by DinB when it is not needed.…”

Section: Tls Polymerasesmentioning

confidence: 99%

The SOS system: A complex and tightly regulated response to DNA damage

Masłowska

Makiela‐Dzbenska

Fijałkowska

2019

Environ and Mol Mutagen

297

212

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Genomes of all living organisms are constantly threatened by endogenous and exogenous agents that challenge the chemical integrity of DNA. Most bacteria have evolved a coordinated response to DNA damage. In Escherichia coli , this inducible system is termed the SOS response. The SOS global regulatory network consists of multiple factors promoting the integrity of DNA as well as error‐prone factors allowing for survival and continuous replication upon extensive DNA damage at the cost of elevated mutagenesis. Due to its mutagenic potential, the SOS response is subject to elaborate regulatory control involving not only transcriptional derepression, but also post‐translational activation, and inhibition. This review summarizes current knowledge about the molecular mechanism of the SOS response induction and progression and its consequences for genome stability. Environ. Mol. Mutagen. 60:368–384, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

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RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

Cited by 8 publications

References 38 publications

Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology

Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology

The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements

The SOS system: A complex and tightly regulated response to DNA damage

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