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Holliday junction

Sagar Khavnekar, Sarath Chandra Dantu, Svetlana Sedelnikova, Sylvia Ayora, John Rafferty, Avinash Kale
Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability...
November 29, 2016: Nucleic Acids Research
Philipp C Nickels, Bettina Wünsch, Phil Holzmeister, Wooli Bae, Luisa M Kneer, Dina Grohmann, Philip Tinnefeld, Tim Liedl
Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer...
October 21, 2016: Science
Mariola R Chacón, Petrina Delivani, Iva M Tolić
Pairing of homologous chromosomes is a crucial step in meiosis, which in fission yeast depends on nuclear oscillations. However, how nuclear oscillations help pairing is unknown. Here, we show that homologous loci typically pair when the spindle pole body is at the cell pole and the nucleus is elongated, whereas they unpair when the spindle pole body is in the cell center and the nucleus is round. Inhibition of oscillations demonstrated that movement is required for initial pairing and that prolonged association of loci leads to mis-segregation...
November 1, 2016: Cell Reports
Peter E Burby, Lyle A Simmons
: Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair pathway (MMR). In contrast, to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Gram-positive bacterium Bacillus subtilis In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B...
October 31, 2016: Journal of Bacteriology
Gizem Ölmezer, Maryna Levikova, Dominique Klein, Benoît Falquet, Gabriele Alessandro Fontana, Petr Cejka, Ulrich Rass
Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2...
October 25, 2016: Nature Communications
Dmitry M Korzhnev, Dante Neculai, Sirano Dhe-Paganon, Cheryl H Arrowsmith, Irina Bezsonova
HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities...
October 22, 2016: Journal of Biomolecular NMR
Getachew Tesfaye Beyene, Seetha V Balasingham, Stephan A Frye, Amine Namouchi, Håvard Homberset, Shewit Kalayou, Tahira Riaz, Tone Tønjum
Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm...
2016: PloS One
Leena Ackermann, Michael Schell, Wojciech Pokrzywa, Éva Kevei, Anton Gartner, Björn Schumacher, Thorsten Hoppe
Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome...
September 26, 2016: Nature Structural & Molecular Biology
Ning Cheng, Yih-Shan Lo, Mohammad Israil Ansari, Kuo-Chieh Ho, Shih-Tong Jeng, Na-Sheng Lin, Hwa Dai
The currently accepted model of recombination-dependent replication (RDR) in plant mitochondrial DNA (mtDNA) does not clearly explain how RDR progresses and how highly complex mtDNA develops. This study aimed to investigate the correlation between RDR and mtDNA complexity during mitochondrial development in mung bean (Vigna radiata) seed, and the initiation and processing of RDR in plant mitochondria. Flow cytometry, pulsed-field gel electrophoresis, electron microscopy, real-time PCR and biochemical studies were used in this study...
September 9, 2016: New Phytologist
Tetsuya Suzuki, Manabu Yasui, Masamitsu Honma
Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. To investigate the mutator phenotype and genetic consequences of DNA double-strand breaks (DSBs) in BS cells, we developed BLM helicase-deficient human cells by disrupting the BLM gene. Cells with a loss of heterozygosity (LOH) due to homologous recombination (HR) or nonhomologous end joining (NHEJ) can be restored with or without site-directed DSB induction. BLM cells exhibited a high frequency of spontaneous interallelic HR with crossover, but noncrossover events with long-tract gene conversions also occurred...
December 1, 2016: Molecular and Cellular Biology
Chetan C Rawal, Sara Riccardo, Chiara Pesenti, Matteo Ferrari, Federica Marini, Achille Pellicioli
Polo-like kinases (PLKs) control several aspects of eukaryotic cell division and DNA damage response. Remarkably, PLKs are overexpressed in several types of cancer, being therefore a marker of bad prognosis. As such, specific PLK kinase activity inhibitors are already used in clinical trials and the regulation of PLK activation is a relevant topic of cancer research. Phosphorylation of threonine residues in the T-loop of the kinase domain is pivotal for PLKs activation. Here, we show that T238A substitution in the T-loop reduces the kinase activity of Cdc5, the only PLK in Saccharomyces cerevisiae, with minor effect on cell growth in unperturbed conditions...
November 2016: Cell Cycle
Dylan J McClung, Abigail Calixto, Mackenzie N Mosera, Raagni Kumar, Ellen L Neidle, Kathryn T Elliott
Despite its presence in most bacteria, yqgF remains one of only 13 essential genes of unknown function in Escherichia coli. Predictions of YqgF function often derive from sequence similarity to RuvC, the canonical Holliday junction resolvase. To clarify its role, we deleted yqgF from a bacterium where it is not essential, Acinetobacter baylyi ADP1. Loss of yqgF impaired growth and increased the frequency of transformation and allelic replacement (TAR). When E. coli yqgF was inserted in place of its A. baylyi chromosomal orthologue, wild-type growth and TAR were restored...
October 2016: Microbiology
Daniela Kobbe, Andy Kahles, Maria Walter, Tobias Klemm, Anja Mannuss, Alexander Knoll, Manfred Focke, Holger Puchta
DNA lesions such as crosslinks represent obstacles for the replication machinery. Nonetheless, replication can proceed via the DNA damage tolerance pathway also known as postreplicative repair pathway. SNF2 ATPase Rad5 homologs, such as RAD5A of the model plant Arabidopsis thaliana, are important for the error-free mode of this pathway. We able to demonstrate before, that RAD5A is a key factor in the repair of DNA crosslinks in Arabidopsis. Here, we show by in vitro analysis that AtRAD5A protein is a DNA translocase able to catalyze fork regression...
July 26, 2016: Plant Journal: for Cell and Molecular Biology
Amandeep Singh, Raghu Bhagavat, M Vijayan, Nagasuma Chandra
In prokaryotes, repair by homologous recombination provides a major means to reinstate the genetic information lost in DNA damage. Recombination repair pathway in mycobacteria has multiple differences as compared to that in Escherichia coli. Of about 20 proteins known to be involved in the pathway, a set of 9 proteins, namely, RecF, RecO, RecR, RecA, SSBa, RuvA, RuvB and RuvC was found to be indispensable among the 43 mycobacterial strains. A domain level analysis indicated that most domains involved in recombination repair are unique to these proteins and are present as single copies in the genomes...
July 2016: Tuberculosis
Soochul Shin, Jinwoo Lee, Sangwoon Yoo, Tomasz Kulikowicz, Vilhelm A Bohr, Byungchan Ahn, Sungchul Hohng
The reactivation of stalled DNA replication via fork regression invokes Holliday junction formation, branch migration, and the recovery of the replication fork after DNA repair or error-free DNA synthesis. The coordination mechanism for these DNA structural transitions by molecular motors, however, remains unclear. Here we perform single-molecule fluorescence experiments with Werner syndrome protein (WRN) and model replication forks. The Holliday junction is readily formed once the lagging arm is unwound, and migrated unidirectionally with 3...
August 2, 2016: Structure
Grégory Hoff, Claire Bertrand, Emilie Piotrowski, Annabelle Thibessard, Pierre Leblond
Most bacterial organisms rely on homologous recombination to repair DNA double-strand breaks and for the post-replicative repair of DNA single-strand gaps. Homologous recombination can be divided into three steps: (i) a pre-synaptic step in which the DNA 3'-OH ends are processed, (ii) a recA-dependent synaptic step allowing the invasion of an intact copy and the formation of Holliday junctions, and (iii) a post-synaptic step consisting of migration and resolution of these junctions. Currently, little is known about factors involved in homologous recombination, especially for the post-synaptic step...
July 14, 2016: Research in Microbiology
Adam J Kolakowski, Jeffrey F Gardner
DOT Integrase (IntDOT) is a member of the tyrosine recombinase family. It catalyzes the integration and excision reactions of an integrative and conjugative element (ICE) called CTnDOT. Like other tyrosine recombinases, the integration reaction proceeds by two sets of strand exchanges between the attDOT site on CTnDOT and an attB site in the host chromosome. The strand exchanges occur seven bases apart and define an overlap region. After the first strand exchanges a Holliday Junction (HJ) intermediate is formed...
July 12, 2016: Plasmid
Prakash Shrestha, Tomoko Emura, Deepak Koirala, Yunxi Cui, Kumi Hidaka, William J Maximuck, Masayuki Endo, Hiroshi Sugiyama, Hanbin Mao
DNA nanoassemblies have demonstrated wide applications in various fields including nanomaterials, drug delivery and biosensing. In DNA origami, single-stranded DNA template is shaped into desired nanostructure by DNA staples that form Holliday junctions with the template. Limited by current methodologies, however, mechanical properties of DNA origami structures have not been adequately characterized, which hinders further applications of these materials. Using laser tweezers, here, we have described two mechanical properties of DNA nanoassemblies represented by DNA nanotubes, DNA nanopyramids and DNA nanotiles...
August 19, 2016: Nucleic Acids Research
Alasdair D J Freeman, Michael Stevens, Anne-Cecile Declais, Adam Leahy, Katherine Mackay, Hassane El Mkami, David M J Lilley, David G Norman
The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process...
August 2, 2016: Biochemistry
Xiaowen Wang, Herui Wang, Bin Guo, Ya Zhang, Yinv Gong, Chi Zhang, Hong Xu, Xiaohui Wu
Resolution of the Holliday junction (HJ) is essential for homologous recombination and DNA repair. In Saccharomyces cerevisiae, HJ resolvase Yen1 and the Mus81-Mms4 complex are redundant in DNA damage repair. In cultured mammalian cells, such redundancy also exists between Yen1 ortholog GEN1 and the Mus81-Mms1 ortholog MUS81-EME1. In this report, we further tested if GEN1 and EME1 redundantly affect HJ-related physiological processes in mice. We found that combined homozygous mutations of Gen1 and Eme1 led to synthetic lethality during early embryonic stages...
July 6, 2016: DNA and Cell Biology
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