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https://www.readbyqxmd.com/read/28475211/short-dna-containing-%C3%AF-sites-enhances-dna-stability-and-gene-expression-in-e-coli-cell-free-transcription-translation-systems
#1
Ryan Marshall, Colin S Maxwell, Scott P Collins, Chase L Beisel, Vincent Noireaux
Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles...
May 5, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28369478/a-non-catalytic-role-of-recbcd-in-homology-directed-gap-repair-and-translesion-synthesis
#2
Luisa Laureti, Lara Lee, Gaëlle Philippin, Vincent Pagès
The RecBCD complex is a key factor in DNA metabolism. This protein complex harbors a processive nuclease and two helicases activities that give it the ability to process duplex DNA ends. These enzymatic activities make RecBCD a major player in double strand break repair, conjugational recombination and degradation of linear DNA. In this work, we unravel a new role of the RecBCD complex in the processing of DNA single-strand gaps that are generated at DNA replication-blocking lesions. We show that independently of its nuclease or helicase activities, the entire RecBCD complex is required for recombinational repair of the gap and efficient translesion synthesis...
June 2, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28202049/costs-and-benefits-of-natural-transformation-in-acinetobacter-baylyi
#3
Nils Hülter, Vidar Sørum, Kristina Borch-Pedersen, Mikkel M Liljegren, Ane L G Utnes, Raul Primicerio, Klaus Harms, Pål J Johnsen
BACKGROUND: Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells. RESULTS: We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions...
February 15, 2017: BMC Microbiology
https://www.readbyqxmd.com/read/28009252/structural-basis-for-the-inhibition-of-recbcd-by-gam-and-its-synergistic-antibacterial-effect-with-quinolones
#4
Martin Wilkinson, Lucy Troman, Wan Ak Wan Nur Ismah, Yuriy Chaban, Matthew B Avison, Mark S Dillingham, Dale B Wigley
Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate...
December 23, 2016: ELife
https://www.readbyqxmd.com/read/27999939/heat-survival-and-phenotype-microarray-profiling-of-salmonella-typhimurium-mutants
#5
Turki M Dawoud, Anita Khatiwara, Si Hong Park, Morgan L Davis, Christopher A Baker, Steven C Ricke, Young Min Kwon
Contamination of food products by pathogenic microorganisms continues to be a major public health and food industry concern. Non-typhoidal Salmonella species have led to numerous outbreaks associated with various foods. A wide variety of methods have been applied and introduced for treatment of fresh foods to eliminate pathogenic as well as spoilage microorganisms. Salmonella can become exposed to elevated temperatures while associated with hosts such as poultry. In addition, heat treatment is also applied at various stages of processing to retain the shelf life of food products...
February 2017: Current Microbiology
https://www.readbyqxmd.com/read/27899634/homologous-recombination-mediated-by-the-mycobacterial-adnab-helicase-without-end-resection-by-the-adnab-nucleases
#6
Richa Gupta, Mihaela-Carmen Unciuleac, Stewart Shuman, Michael S Glickman
Current models of bacterial homologous recombination (HR) posit that extensive resection of a DNA double-strand break (DSB) by a multisubunit helicase-nuclease machine (e.g. RecBCD, AddAB or AdnAB) generates the requisite 3' single-strand DNA substrate for RecA-mediated strand invasion. AdnAB, the helicase-nuclease implicated in mycobacterial HR, consists of two subunits, AdnA and AdnB, each composed of an N-terminal ATPase domain and a C-terminal nuclease domain. DSB unwinding by AdnAB in vitro is stringently dependent on the ATPase activity of the 'lead' AdnB motor translocating on the 3' ssDNA strand, but not on the putative 'lagging' AdnA ATPase...
January 25, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/27881980/comparative-genomics-of-dna-recombination-and-repair-in-cyanobacteria-biotechnological-implications
#7
REVIEW
Corinne Cassier-Chauvat, Théo Veaudor, Franck Chauvat
Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product...
2016: Frontiers in Microbiology
https://www.readbyqxmd.com/read/27644322/mechanism-for-nuclease-regulation-in-recbcd
#8
Martin Wilkinson, Yuriy Chaban, Dale B Wigley
In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit...
September 20, 2016: ELife
https://www.readbyqxmd.com/read/27607601/correction-to-the-nuclease-domain-of-the-escherichia-coli-recbcd-enzyme-catalyzes-degradation-of-linear-and-circular-single-stranded-and-double-stranded-dna
#9
Jian-Zhong Sun, Douglas A Julin, Jin-Shan Hu
No abstract text is available yet for this article.
September 20, 2016: Biochemistry
https://www.readbyqxmd.com/read/27422010/processive-dna-unwinding-by-recbcd-helicase-in-the-absence-of-canonical-motor-translocation
#10
Michael J Simon, Joshua E Sokoloski, Linxuan Hao, Elizabeth Weiland, Timothy M Lohman
Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors...
July 31, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/27401752/recbcd-enzyme-chi-recognition-mutants-recognize-chi-recombination-hotspots-in-the-right-dna-context
#11
Susan K Amundsen, Jake W Sharp, Gerald R Smith
RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC...
September 2016: Genetics
https://www.readbyqxmd.com/read/27330137/unexpected-dna-context-dependence-identifies-a-new-determinant-of-chi-recombination-hotspots
#12
Andrew F Taylor, Susan K Amundsen, Gerald R Smith
Homologous recombination occurs especially frequently near special chromosomal sites called hotspots. In Escherichia coli, Chi hotspots control RecBCD enzyme, a protein machine essential for the major pathway of DNA break-repair and recombination. RecBCD generates recombinogenic single-stranded DNA ends by unwinding DNA and cutting it a few nucleotides to the 3' side of 5' GCTGGTGG 3', the sequence historically equated with Chi. To test if sequence context affects Chi activity, we deep-sequenced the products of a DNA library containing 10 random base-pairs on each side of the Chi sequence and cut by purified RecBCD...
September 30, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27220465/sequence-dependent-nanometer-scale-conformational-dynamics-of-individual-recbcd-dna-complexes
#13
Ashley R Carter, Maasa H Seaberg, Hsiu-Fang Fan, Gang Sun, Christopher J Wilds, Hung-Wen Li, Thomas T Perkins
RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2-4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD-DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex...
July 8, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/26904381/pre-early-functions-of-bacteriophage-t5-and-its-relatives
#14
REVIEW
John Davison
Coliphage T5 injects its DNA in 2 steps: the first step transfer (FST) region 7.9% is injected and its genes are expressed and only then does the remainder (second step transfer, SST) of its DNA enter the cell. In the FST region, only 2 essential genes (A1 and A2) have been identified and a third (dmp) non-essential gene codes for a deoxyribonucleotide 5' monophosphatase. Thirteen additional putative ORFs are present in the FST region. Numerous properties have been attributed to FST region, including SST, host DNA degradation, inhibition of host RNA and protein synthesis, restriction insensitivity and protection of T5 DNA...
October 2015: Bacteriophage
https://www.readbyqxmd.com/read/26874520/single-strand-gap-repair-involves-both-recf-and-recbcd-pathways
#15
REVIEW
Vincent Pagès
Homologous recombination repairs discontinuities in DNA including single-strand gaps (SSGs) and double-strand breaks (DSBs). This commentary describes how the RecBCD and RecF pathways might be exchangeable for the repair of their respective DSB and SSG canonical substrates. In particular, I will discuss how the RecBCD pathway could engage in the repair of an SSG even when the latter is not associated with a DSB.
August 2016: Current Genetics
https://www.readbyqxmd.com/read/26762976/analysis-of-lexa-binding-sites-and-transcriptomics-in-response-to-genotoxic-stress-in-leptospira-interrogans
#16
Luciane Schons-Fonseca, Josefa B da Silva, Juliana S Milanez, Renan H Domingos, Janet L Smith, Helder I Nakaya, Alan D Grossman, Paulo L Ho, Renata M A da Costa
We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e...
February 18, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/26471352/inhibition-of-recbcd-in-klebsiella-pneumoniae-by-gam-and-its-effect-on-the-efficiency-of-gene-replacement
#17
Chuan Chen, Dong Wei, Pengfu Liu, Min Wang, Jiping Shi, Biao Jiang, Jian Hao
Gam protein is an inhibitor of the host RecBCD exonuclease, and this inhibition is essential to the proficiency of Red recombinase-mediated gene replacement. In Klebsiella pneumoniae, the efficiency of this gene replacement was lower than that in Escherichia coli, and the minimum length of homologous extensions required was longer. Thus, it was supposed that the inhibitory effect of Gam against RecBCD was weak in K. pneumoniae. To test this hypothesis, a Gam-deficient Red recombinase expression plasmid and a ΔrecB K...
February 2016: Journal of Basic Microbiology
https://www.readbyqxmd.com/read/26261330/quantitative-genomic-analysis-of-reca-protein-binding-during-dna-double-strand-break-repair-reveals-recbcd-action-in-vivo
#18
Charlotte A Cockram, Milana Filatenkova, Vincent Danos, Meriem El Karoui, David R F Leach
Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli...
August 25, 2015: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/26195593/recf-and-recr-play-critical-roles-in-the-homologous-recombination-and-single-strand-annealing-pathways-of-mycobacteria
#19
Richa Gupta, Stewart Shuman, Michael S Glickman
Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro, also participates in the SSA pathway...
October 2015: Journal of Bacteriology
https://www.readbyqxmd.com/read/26003632/recbcd-is-required-to-complete-chromosomal-replication-implications-for-double-strand-break-frequencies-and-repair-mechanisms
#20
REVIEW
Justin Courcelle, Brian M Wendel, Dena D Livingstone, Charmain T Courcelle
Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination...
August 2015: DNA Repair
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