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Microbiology and Molecular Biology... Dec 2008The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear... (Review)
Review
The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5' strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.
Topics: DNA Breaks, Double-Stranded; DNA Repair; DNA, Bacterial; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Recombination, Genetic
PubMed: 19052323
DOI: 10.1128/MMBR.00020-08 -
Communications Biology 2019Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3'-ends (MSD3Es) to establish...
Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3'-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3' termini (about 20% of the total) was not repaired after the removal of 3' phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3'- > 5' exonuclease activity of exonuclease III (3'- > 5' exonuclease, insensitive to the 3' phosphate group) was counterbalanced by the 5'- > 3' polymerase activity of T4DP. This combined reaction, termed "SB-repairing" (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es.
Topics: DNA; DNA Nucleotidylexotransferase; DNA Repair; DNA-Directed DNA Polymerase; Deoxyribonuclease IV (Phage T4-Induced); Exodeoxyribonucleases; Sonication; Substrate Specificity
PubMed: 31728420
DOI: 10.1038/s42003-019-0660-7 -
Current Biology : CB Dec 1997The octameric Chi (chi) sequence is a recombination hotspot in Escherichia coli. Recent studies suggest a singular mechanism by which chi regulates not only the nuclease... (Review)
Review
The octameric Chi (chi) sequence is a recombination hotspot in Escherichia coli. Recent studies suggest a singular mechanism by which chi regulates not only the nuclease activity of RecBCD enzyme, but also the ability of RecBCD to promote loading of the strand exchange protein, RecA, onto chi-containing DNA.
Topics: Binding Sites; DNA, Bacterial; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Mutation; Rec A Recombinases; Recombination, Genetic
PubMed: 9382825
DOI: 10.1016/s0960-9822(06)00394-0 -
Nucleic Acids Research Sep 2020RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ)...
RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ) deletion strain exhibited extreme sensitivity to hydrogen peroxide and methyl-methanesulphonate, as well as a high spontaneous mutation rate and an accumulation of unrepaired abasic sites in vivo, indicating the involvement of drRecJ in the BER pathway. The binding affinity and nuclease activity preference of drRecJ toward DNA substrates containing a 5'-P-dSpacer group, a 5'-deoxyribose-phosphate (dRP) mimic, were established. A 1.9 Å structure of drRecJ in complex with 5'-P-dSpacer-modified single-stranded DNA (ssDNA) revealed a 5'-monophosphate binding pocket and occupancy of 5'-dRP in the drRecJ nuclease core. The mechanism for RecJ 5'-dRP catalysis was explored using structural and biochemical data, and the results implied that drRecJ is not a canonical 5'-dRP lyase. Furthermore, in vitro reconstitution assays indicated that drRecJ tends to participate in the long-patch BER pathway rather than the short-patch BER pathway.
Topics: Bacterial Proteins; Binding Sites; DNA Repair; Deinococcus; Exodeoxyribonucleases; Protein Binding
PubMed: 32870272
DOI: 10.1093/nar/gkaa714 -
Microbiology (Reading, England) Nov 2020Homologous recombination plays key roles in fundamental processes such as recovery from DNA damage and in bacterial horizontal gene transfer, yet there are still open...
Homologous recombination plays key roles in fundamental processes such as recovery from DNA damage and in bacterial horizontal gene transfer, yet there are still open questions about the distribution of recognized components of recombination machinery among bacteria and archaea. RecBCD helicase-nuclease plays a central role in recombination among Gammaproteobacteria like ; while bacteria in other phyla, like the Firmicute , use the related AddAB complex. The activity of at least some of these complexes is controlled by short DNA sequences called crossover hotspot instigator (Chi) sites. When RecBCD or AddAB complexes encounter an autologous Chi site during unwinding, they introduce a nick such that ssDNA with a free end is available to invade another duplex. If homologous DNA is present, RecA-dependent homologous recombination is promoted; if not (or if no autologous Chi site is present) the RecBCD/AddAB complex eventually degrades the DNA. We examined the distribution of and genes among bacteria, and sought ways to distinguish them unambiguously. We examined bacterial species among 33 phyla, finding some unexpected distribution patterns. and are less conserved than , with the orthologous and genes more conserved than the or genes. We were able to classify RecB vs. AddA and RecC vs. AddB in some bacteria where this had not previously been done. We used logo analysis to identify sequence segments that are conserved, but differ between the RecBC and AddAB proteins, to help future differentiation between members of these two families.
Topics: Amino Acid Sequence; Bacteria; Base Sequence; Conserved Sequence; DNA, Bacterial; Evolution, Molecular; Exodeoxyribonuclease V; Exodeoxyribonucleases; Gene Transfer, Horizontal; Phylogeny; Rec A Recombinases; Recombination, Genetic
PubMed: 33085588
DOI: 10.1099/mic.0.000980 -
Proceedings of the National Academy of... Jul 2001Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers... (Review)
Review
Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.
Topics: Adenosine Triphosphatases; Animals; DNA Damage; DNA Repair Enzymes; DNA, Single-Stranded; DNA-Binding Proteins; Eukaryotic Cells; Exodeoxyribonuclease V; Exodeoxyribonucleases; Fungal Proteins; Humans; Meiosis; Mitosis; Rad51 Recombinase; Rec A Recombinases; Recombination, Genetic; Replication Protein A; Saccharomyces cerevisiae Proteins
PubMed: 11459983
DOI: 10.1073/pnas.121046198 -
Analytical Sciences : the International... 2018In this paper, we describe a simple and highly sensitive fluorescence strategy of mercury ions based on exonuclease III (Exo III)-aided target recycling amplification to...
In this paper, we describe a simple and highly sensitive fluorescence strategy of mercury ions based on exonuclease III (Exo III)-aided target recycling amplification to ensure sensitivity. With an ultra high sensitivity (1 pM), our strategy has been simple and cost-effective, which does not need any artificial modification fluorescence groups, and can be carried out in a pot. It also shows excellent selectivity. Therefore, our new method provides an effective platform for mercury-ion detection.
Topics: Biosensing Techniques; DNA Probes; Exodeoxyribonucleases; Limit of Detection; Mercury; Spectrometry, Fluorescence
PubMed: 29526891
DOI: 10.2116/analsci.34.259 -
PLoS Genetics Sep 2021Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA...
Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.
Topics: DNA Breaks, Double-Stranded; DNA-Binding Proteins; Exodeoxyribonucleases; Genes, Fungal; Histones; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34520455
DOI: 10.1371/journal.pgen.1009807 -
Science Advances Jul 2021FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks...
FAN1, a DNA structure-specific nuclease, interacts with MLH1, but the repair pathways in which this complex acts are unknown. FAN1 processes DNA interstrand crosslinks (ICLs) and FAN1 variants are modifiers of the neurodegenerative Huntington's disease (HD), presumably by regulating HD-causing CAG repeat expansions. Here, we identify specific amino acid residues in two adjacent FAN1 motifs that are critical for MLH1 binding. Disruption of the FAN1-MLH1 interaction confers cellular hypersensitivity to ICL damage and defective repair of CAG/CTG slip-outs, intermediates of repeat expansion mutations. FAN1-S126 phosphorylation, which hinders FAN1-MLH1 association, is cell cycle-regulated by cyclin-dependent kinase activity and attenuated upon ICL induction. Our data highlight the FAN1-MLH1 complex as a phosphorylation-regulated determinant of ICL response and repeat stability, opening novel paths to modify cancer and neurodegeneration.
Topics: DNA; DNA Damage; Endodeoxyribonucleases; Exodeoxyribonucleases; Multifunctional Enzymes
PubMed: 34330701
DOI: 10.1126/sciadv.abf7906 -
Genes Oct 2019Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation,...
Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders' genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking and but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.
Topics: Adaptation, Physiological; CRISPR-Cas Systems; DNA Repair; Escherichia coli; Escherichia coli Proteins; Exodeoxyribonuclease V; Exodeoxyribonucleases; Exonucleases; Genome, Bacterial; Genomic Instability
PubMed: 31683605
DOI: 10.3390/genes10110872