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Isidoro Feliciello, Davor Zahradka, Ksenija Zahradka, Siniša Ivanković, Nikolina Puc, Damir Đermić
Double strand breaks (DSBs) in E. coli chromosome (such as those induced by gamma rays) are repaired by recombination repair, during which a certain amount of DNA gets degraded. We monitored DNA degradation in gamma-irradiated cells to assess processing of DSBs. DNA degradation in irradiated cells is regulated by RecA protein concentration and its affinity of ssDNA binding, as well as by exonucleases that trim 3'-terminated ss tails. Here we determined the effects of proteins that affect formation and stability of RecA nucleofilaments on DNA degradation and cell survival...
March 16, 2018: Biochimie
Amandeep Singh, M Vijayan, Umesh Varshney
In addition to the canonical Single Stranded DNA Binding (SSBa) protein, many bacterial species, including mycobacteria, have a paralogous SSBb. The SSBb proteins have not been well characterized. While in B. subtilis, SSBb has been shown to be involved in genetic recombination; in S. coelicolor it mediates chromosomal segregation during sporulation. Sequence analysis of SSBs from mycobacterial species suggests low conservation of SSBb proteins, as compared to the conservation of SSBa proteins. Like most bacterial SSB proteins, M...
January 2018: Tuberculosis
Hung-Yi Wu, Chih-Hao Lu, Hung-Wen Li
E. coli RecA recombinase catalyzes the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments, as the first step of this repair process. It has been suggested that RecA filaments assemble mainly by binding and extending onto the free ssDNA region not covered by SSB, or are assisted by mediators. Using the tethered particle motion (TPM) technique, we monitored individual RecA filament assembly on SSB-wrapped ssDNA in real-time...
September 19, 2017: Scientific Reports
Aman Kumar Ujaoney, Mahesh Kumar Padwal, Bhakti Basu
Deinococcus radiodurans is inherently resistant to both ionizing radiation and desiccation. Fifteen months of desiccation was found to be the LD50 dose for D. radiodurans. Desiccated cells of D. radiodurans entered 6h of growth arrest during post-desiccation recovery (PDR). Proteome dynamics during PDR were mapped by resolving cellular proteins by 2-dimensional gel electrophoresis coupled with mass spectrometry. At least 41 proteins, represented by 51 spots on proteome profiles, were differentially expressed throughout PDR...
September 2017: Biochimica et Biophysica Acta
Hui Yin Tan, Luke A Wilczek, Sasheen Pottinger, Maria Manosas, Cong Yu, Trong Nguyenduc, Piero R Bianco
The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB-interactome. Key to both roles is the C-terminal, one-third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target...
April 2017: Protein Science: a Publication of the Protein Society
S Korolev
Recombination mediator proteins (RMPs) are critical for genome integrity in all organisms. They include phage UvsY, prokaryotic RecF, -O, -R (RecFOR) and eukaryotic Rad52, Breast Cancer susceptibility 2 (BRCA2) and Partner and localizer of BRCA2 (PALB2) proteins. BRCA2 and PALB2 are tumor suppressors implicated in cancer. RMPs regulate binding of RecA-like recombinases to sites of DNA damage to initiate the most efficient non-mutagenic repair of broken chromosome and other deleterious DNA lesions. Mechanistically, RMPs stimulate a single-stranded DNA (ssDNA) hand-off from ssDNA binding proteins (ssbs) such as gp32, SSB and RPA, to recombinases, activating DNA repair only at the time and site of the damage event...
June 2017: Biophysical Chemistry
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
Laure Lavatine, Susu He, Anne Caumont-Sarcos, Catherine Guynet, Brigitte Marty, Mick Chandler, Bao Ton-Hoang
Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the 'peel and paste' pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork...
September 19, 2016: Nucleic Acids Research
Wellington C Leite, Carolina W Galvão, Sérgio C Saab, Jorge Iulek, Rafael M Etto, Maria B R Steffens, Sindhu Chitteni-Pattu, Tyler Stanage, James L Keck, Michael M Cox
The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins...
2016: PloS One
Sukrit Suksombat, Rustem Khafizov, Alexander G Kozlov, Timothy M Lohman, Yann R Chemla
Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated...
August 25, 2015: ELife
Roshan Singh Thakur, Shivakumar Basavaraju, Jasbeer Singh Khanduja, K Muniyappa, Ganesh Nagaraju
Aberrant DNA replication, defects in the protection, and restart of stalled replication forks are major causes of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB, and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen, responds to different types of replication stress and DNA damage are unclear...
October 2, 2015: Journal of Biological Chemistry
Taejin Kim, Sindhu Chitteni-Pattu, Benjamin L Cox, Elizabeth A Wood, Steven J Sandler, Michael M Cox
The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis...
June 2015: PLoS Genetics
Shimin Le, Mingxi Yao, Jin Chen, Artem K Efremov, Sara Azimi, Jie Yan
Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions-diameter (D): 40-50 μm, height (H): 100 μm; H:D∼2:1)...
September 30, 2015: Nucleic Acids Research
Aman K Ujaoney, Bhakti Basu, K Muniyappa, Shree K Apte
Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N-terminal without connector), SsbNC (N-terminal with connector) and SsbC (C-terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (SsbFL)...
2015: FEBS Open Bio
Mónica Fernández-Sierra, Edwin Quiñones
Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect...
March 15, 2015: Archives of Biochemistry and Biophysics
Katsumi Morimatsu, Stephen C Kowalczykowski
Recombinational DNA repair by the RecF pathway of Escherichia coli requires the coordinated activities of RecA, RecFOR, RecQ, RecJ, and single-strand DNA binding (SSB) proteins. These proteins facilitate formation of homologously paired joint molecules between linear double-stranded (dsDNA) and supercoiled DNA. Repair starts with resection of the broken dsDNA by RecQ, a 3'→5' helicase, RecJ, a 5'→3' exonuclease, and SSB protein. The ends of a dsDNA break can be blunt-ended, or they may possess either 5'- or 3'-single-stranded DNA (ssDNA) overhangs of undefined length...
December 2, 2014: Proceedings of the National Academy of Sciences of the United States of America
Sankalp Gupta, Joseph T P Yeeles, Kenneth J Marians
Stalled replication forks are sites of chromosome breakage and the formation of toxic recombination intermediates that undermine genomic stability. Thus, replication fork repair and reactivation are essential processes. Among the many models of replication fork reactivation is one that invokes fork regression catalyzed by the strand exchange protein RecA as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecA using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage...
October 10, 2014: Journal of Biological Chemistry
Fiona A Curtis, Ali D Malay, Alexander J Trotter, Lindsay A Wilson, Michael M H Barradell-Black, Laura Y Bowers, Patricia Reed, Christopher R T Hillyar, Robert P Yeo, John M Sanderson, Jonathan G Heddle, Gary J Sharples
Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized...
2014: PloS One
Justin S Lenhart, Eileen R Brandes, Jeremy W Schroeder, Roderick J Sorenson, Hollis D Showalter, Lyle A Simmons
RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair...
August 2014: Journal of Bacteriology
Kaiying Cheng, Xin Xu, Ye Zhao, Liangyan Wang, Guangzhi Xu, Yuejin Hua
The RecFOR DNA repair pathway is one of the major RecA-dependent recombinatorial repair pathways in bacteria and plays an important role in double-strand breaks repair. RecO, one of the major recombination mediator proteins in the RecFOR pathway, has been shown to assist RecA loading onto single-stranded binding protein (SSB) coated single-stranded DNA (ssDNA). However, it has not been characterized whether the protein-protein interaction between RecO and SSB contributes to that process in vivo. Here, we identified the residue arginine-121 of Deinococcus radiodurans RecO (drRecO-R121) as the key residue for RecO-SSB interaction...
May 2014: Acta Biochimica et Biophysica Sinica
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