The DNA Exonucleases of
            <i>Escherichia coli</i>

Lovett, Susan

doi:10.1128/ecosalplus.4.4.7

Cited by 91 publications

(76 citation statements)

References 298 publications

(307 reference statements)

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“…The products of the xseA, xonA and sbcDC genes (exonuclease VII, exonuclease I and SbcCD exo/endonuclease respectively) are the major 3 0 -5 0 exonucleases in E. coli. They participate in several DNA repair and genome stability pathways and the reader is directed to review [110] for a more detailed discussion of their functions. The over-replication in the xseA xonA sbcDC mutant is very interesting and does indeed suggest the existence of a pathway of DNA amplification involving 3 0 overhangs.…”

Section: Recg Controls Dna Amplification During Dsbr and At Arrested mentioning

confidence: 99%

RecG controls DNA amplification at double‐strand breaks and arrested replication forks

Azeroglu

Leach

2017

FEBS Letters

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DNA amplification is a powerful mutational mechanism that is a hallmark of cancer and drug resistance. It is therefore important to understand the fundamental pathways that cells employ to avoid over‐replicating sections of their genomes. Recent studies demonstrate that, in the absence of RecG, DNA amplification is observed at sites of DNA double‐strand break repair (DSBR) and of DNA replication arrest that are processed to generate double‐strand ends. RecG also plays a role in stabilising joint molecules formed during DSBR. We propose that RecG prevents a previously unrecognised mechanism of DNA amplification that we call reverse‐restart, which generates DNA double‐strand ends from incorrect loading of the replicative helicase at D‐loops formed by recombination, and at arrested replication forks.

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Section: Recg Controls Dna Amplification During Dsbr and At Arrested mentioning

confidence: 99%

RecG controls DNA amplification at double‐strand breaks and arrested replication forks

Azeroglu

Leach

2017

FEBS Letters

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“…Both ExoI and ExoVII are strongly specific for ssDNA (reviewed in [30]) and may therefore function in the cell to scavenge unwound 3′ strands from the replication fork, a likely intermediate during the template-switch events. ExoI is in the DnaQ family of exonucleases, some of which are associated with DNA polymerases as “proofreading” functions.…”

Section: Resultsmentioning

confidence: 99%

SSB recruitment of Exonuclease I aborts template-switching in Escherichia coli

et al. 2017

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Misalignment of a nascent strand and the use of an alternative template during DNA replication, a process termed “template-switching”, can give rise to frequent mutations and genetic rearrangements. Mutational hotspots are frequently found associated with imperfect inverted repeats (“quasipalindromes” or “QPs”) in many organisms, including bacteriophage, bacteria, yeast and mammals. Evidence suggests that QPs mutate by a replication template-switch whereby one copy of the inverted repeat templates synthesis of the other. To study quasipalindrome-associated mutagenesis (“QPM”) more systematically, we have engineered mutational reporters in the lacZ gene of Escherichia coli, that revert to Lac+ specifically by QPM. We and others have shown that QPM is more efficient during replication of the leading strand than it is on the lagging strand. We have previously shown that QPM is elevated and that the leading-strand bias is lost in mutants lacking the major 3′ ssDNA exonucleases, ExoI and ExoVII. This suggests that one or both of these exonucleases more efficiently abort template-switches on the lagging strand. Here, we show that ExoI is primarily responsible for this bias and that its ability to be recruited by single-strand DNA binding protein plays a critical role in QPM avoidance and strand bias. In addition to these stand-alone exonucleases, loss of the 3′ proofreading exonuclease activity of the replicative DNA polymerase III also greatly elevates QPM. This may be because template-switching is initiated by base misincorporation, leading to polymerase dissociation and subsequent nascent strand misalignment; alternatively or additionally, the proofreading exonuclease may scavenge displaced 3′ DNA that would otherwise be free to misalign.

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“…However, in the approach used here, maintenance of the f1 origin and the selection marker in the produced phage allows for reinfection across the culture, which is important for subsequent biological amplification such as needed in phage display and, here, archival information storage. Moreover, circularization blocks exonuclease activity, which may prove important for therapeutic applications (41). In the future, improved understanding of phage biology should enable new approaches to excising specific coding sequences from M13 to generate engineered systems specifically designed for production of cssDNA.…”

Section: Discussionmentioning

confidence: 99%

Bioproduction of single-stranded DNA from isogenic miniphage

Shepherd

Huang

et al. 2019

Preprint

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Scalable production of gene-length single-stranded DNA (ssDNA) with sequence control has applications in homology directed repair templating, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular single-stranded DNA (cssDNA) using bacteriophage M13 addresses these needs at low cost. A primary goal toward this end is to minimize the essential protein coding regions of the produced, exported sequence while maintaining its infectivity and production purity, with engineered regions of sequence control. Synthetic miniphage constitutes an ideal platform for bacterial production of isogenic cssDNA, using inserts of custom sequence and size to attain this goal, offering an inexpensive resource at milligram and higher synthesis scales. Here, we show that the Escherichia coli (E. coli) helper strain M13cp combined with a miniphage genome carrying only an f1 origin and a β-lactamase-encoding (bla) antibiotic resistance gene enables the production of pure cssDNA with a minimum sequence genomic length of 1,676 nt directly from bacteria, without the need for additional purification from contaminating dsDNA, genomic DNA, or fragmented DNAs. Low-cost scalability of isogenic, custom-length cssDNA is also demonstrated for a sequence of 2,520 nt using a commercial bioreactor. We apply this system to generate cssDNA for the programmed self-assembly of wireframe DNA origami objects with exonucleaseresistant, custom-designed circular scaffolds that are purified with low endotoxin levels (<5 E.U./ml) for therapeutic applications. We also encode digital information that is stored on the genome with application to write-once, read-many archival data storage.

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The DNA Exonucleases of Escherichia coli

Cited by 91 publications

References 298 publications

RecG controls DNA amplification at double‐strand breaks and arrested replication forks

RecG controls DNA amplification at double‐strand breaks and arrested replication forks

SSB recruitment of Exonuclease I aborts template-switching in Escherichia coli

Bioproduction of single-stranded DNA from isogenic miniphage

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