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single-stranded DNA binding protein

Yan Zeng, Yang Cui, Yong Zhang, Yanruo Zhang, Meng Liang, Hui Chen, Jie Lan, Guangtao Song, Jizhong Lou
CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique...
November 14, 2017: Nucleic Acids Research
Tomáš Špaček, Vojtěch Pavluch, Lukáš Alán, Nikola Capková, Hana Engstová, Andrea Dlasková, Zuzana Berková, František Saudek, Petr Ježek
Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry...
November 15, 2017: Scientific Reports
Elina Cernooka, Janis Rumnieks, Kaspars Tars, Andris Kazaks
Modern DNA sequencing capabilities have led to the discovery of a large number of new bacteriophage genomes, which are a rich source of novel proteins with an unidentified biological role. The genome of Enterobacter cancerogenus bacteriophage Enc34 contains several proteins of unknown function that are nevertheless conserved among distantly related phages. Here, we report the crystal structure of a conserved Enc34 replication protein ORF6 which contains a domain of unknown function DUF2815. Despite the low (~15%) sequence identity, the Enc34 ORF6 structurally resembles the gene 2...
November 14, 2017: Scientific Reports
Li Gao, Qin Li, Zebin Deng, Brendan Brady, Ni Xia, Yang Zhou, Haixia Shi
Molybdenum disulfide (MoS2) has shown highly attractive superiority as a platform for sensing. However, DNA physisorption on the surface of MoS2 was susceptible to nonspecific probe displacement and false-positive signals. To solve these problems, we have developed a novel MoS2-aptamer nanosheet biosensor for detecting thrombin using a covalently linked aptamer to the MoS2 nanosheet. Ten percent Tween 80 was used to prevent thrombin from nonspecific binding and to rapidly form thiol-DNA/gold nanoparticle (AuNP) conjugates...
2017: International Journal of Nanomedicine
Thomas Wiegand, Wei-Chih Liao, Ta Chung Ong, Alexander Däpp, Riccardo Cadalbert, Christophe Copéret, Anja Böckmann, Beat H Meier
DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein-DNA and protein-ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein-DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy...
November 8, 2017: Journal of Biomolecular NMR
Goutam Mukherjee, Arumay Pal, Yaakov Levy
In prokaryotes, the RecA protein catalyzes the repair and strand exchange of double-stranded DNA. RecA binds to single-stranded DNA (ssDNA) and forms a presynaptic complex in which the protein polymerizes around the ssDNA to form a right-handed helical nucleoprotein filament structure. In the present work, the mechanism for the formation of the RecA-ssDNA filament structure is modeled using coarse-grained molecular dynamics simulations. Information from the X-ray structure was used to model the protein itself but not its interactions; the interactions between the protein and the ssDNA were modeled solely by electrostatic, aromatic, and repulsive energies...
November 6, 2017: Molecular BioSystems
Tatiana A Nevzorova, Qingze Zhao, Yakov A Lomakin, Anastasia A Ponomareva, Alexander R Mukhitov, Prashant K Purohit, John W Weisel, Rustem I Litvinov
Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibodycoated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface...
March 2017: BioNanoScience
Michelle C Silva, Katie E Bryan, Milagros D Morrical, April M Averill, Julie Dragon, Adrian P Wiegmans, Scott W Morrical
The human RAD51 recombinase possesses DNA pairing and strand exchange activities that are essential for the error-free, homology-directed repair of DNA double-strand breaks. The recombination activities of RAD51 are activated upon its assembly into presynaptic filaments on single-stranded DNA at resected DSB ends. Defects in filament assembly caused by mutations in RAD51 or its regulators such as BRCA2 are associated with human cancer. Here we describe two novel RAD51 missense variants located in the multimerization/BRCA2 binding region of RAD51...
October 23, 2017: DNA Repair
Julie K Horton, Donna F Stefanick, Ming-Lang Zhao, Agnes K Janoshazi, Natalie R Gassman, Hannah J Seddon, Samuel H Wilson
Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3'-phosphate and 5'-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3'-OH and 5'-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage...
October 19, 2017: DNA Repair
Junbo Chen, Albert Zuehlke, Bin Deng, Hanyong Peng, Xiandeng Hou, Hongquan Zhang
We report here the concept of a self-powered, target-triggered DNA motor constructed by engineering a DNAzyme to adapt into binding-induced DNA assembly. An affinity ligand was attached to the DNAzyme motor via a DNA spacer, and a second affinity ligand was conjugated to the gold nanoparticle (AuNP) that was also decorated with hundreds of substrate strands serving as a high-density, three-dimensional track for the DNAzyme motor. Binding of a target molecule to the two ligands induced hybridization between the DNAzyme and its substrate on the AuNP, which are otherwise unable to spontaneously hybridize...
November 15, 2017: Analytical Chemistry
Shuang Wang, Shasha Lu, Jiahui Zhao, Jianshe Huang, Xiurong Yang
G-quadruplex plays roles in numerous physiological and pathological processes of organisms. Due to the unique properties of G-quadruplex (e.g., forming G4/hemin complexes with catalytic activity and electron acceptability, binding with metal ions, proteins, fluorescent ligands, and so on), it has been widely applied in biosensing. But the formation process of G-quadruplex is not yet fully understood. Here, a DNA tetrahedron platform with higher reproducibility, regenerative ability, and time-saving building process was coupled with dual polarization interferometry technique for the real-time and label-free investigation of the specific interaction process of guanine-rich singled-stranded DNA (G-rich ssDNA) and Pb(2+)...
November 14, 2017: ACS Applied Materials & Interfaces
Chih-Hao Lu, Ting-Tzu Chang, Chia-Chuan Cho, Hui-Cin Lin, Hung-Wen Li
RecA plays central roles in the homologous recombination to repair double-stranded DNA break damage in E. coli. A previously identified recA strain surviving high doses of UV radiation includes a dominant RecA E38K mutation. Using single-molecule experiments, we showed that the RecA E38K variant protein assembles nucleoprotein filaments more rapidly than the wild-type RecA. We also used a single-molecule fluorescence resonance energy transfer (smFRET) experiment to compare the nucleation cluster dynamics of wild-type RecA and RecA E38K mutants on various short ssDNA substrates...
November 2, 2017: Scientific Reports
Megan D Warner, Ishara F Azmi, Yanding Zhao, Sukhyun Kang, Stephen P Bell
In eukaryotes, DNA replication initiates from multiple origins of replication for timely genome duplication. These sites are selected by origin licensing, during which the core enzyme of the eukaryotic DNA replicative helicase, the Mcm2-7 (minichromosome maintenance) complex, is loaded at each origin. This origin licensing requires loading two Mcm2-7 helicases around origin DNA in a head-to-head orientation. Current models suggest that the origin recognition complex (ORC) and cell-division cycle 6 (Cdc6) proteins recognize and encircle origin DNA and assemble an Mcm2-7 double hexamer around adjacent double-stranded DNA...
October 26, 2017: Journal of Biological Chemistry
Ruvini Kariawasam, Maddison Knight, Roland Gamsjaeger, Liza Cubeddu
Single stranded DNA-binding proteins (SSBs) are essential for the maintenance of genome integrity and are required in in all known cellular organisms. Over the last 10 years, the role of two new human SSBs, hSSB1 (NABP2/OBFC2B) and hSSB2 (NABP1/OBFC2A), has been described and characterised in various important DNA repair processes. Both these proteins are made up of a conserved oligonucleotide-binding (OB) fold that is responsible for ssDNA recognition as well a unique flexible carboxy-terminal extension involved in protein-protein interactions...
October 24, 2017: Biomolecular NMR Assignments
Christopher Maffeo, Aleksei Aksimentiev
During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA-SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment...
October 20, 2017: Nucleic Acids Research
Maria Mills, Gábor M Harami, Yeonee Seol, Máté Gyimesi, Máté Martina, Zoltán J Kovács, Mihály Kovács, Keir C Neuman
The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments...
November 16, 2017: Nucleic Acids Research
Gemma S Puts, M Kathryn Leonard, Nidhi V Pamidimukkala, Devin E Snyder, David M Kaetzel
The NME family of proteins is composed of 10 isoforms, designated NME1-10, which are diverse in their enzymatic activities and patterns of subcellular localization. Each contains a conserved domain associated with a nucleoside diphosphate kinase (NDPK) function, although not all are catalytically active. Several of the NME isoforms (NME1, NME5, NME7, and NME8) also exhibit a 3'-5' exonuclease activity, suggesting roles in DNA proofreading and repair. NME1 and NME2 have been shown to translocate to the nucleus, although they lack a canonical nuclear localization signal...
October 23, 2017: Laboratory Investigation; a Journal of Technical Methods and Pathology
Ci Ji Lim, Arthur J Zaug, Hee Jin Kim, Thomas R Cech
The human shelterin proteins associate with telomeric DNA to confer telomere protection and length regulation. They are thought to form higher-order protein complexes for their functions, but studies of shelterin proteins have been mostly limited to pairs of proteins. Here we co-express various human shelterin proteins and find that they form defined multi-subunit complexes. A complex harboring both TRF2 and POT1 has the strongest binding affinity to telomeric DNA substrates comprised of double-stranded DNA with a 3' single-stranded extension...
October 20, 2017: Nature Communications
Raphael Souza Pavani, Marcela O Vitarelli, Carlos A H Fernandes, Fabio F Mattioli, Mariana Morone, Milene C Menezes, Marcos R M Fontes, Maria Isabel N Cano, Maria Carolina Elias
Replication protein A (RPA), the major eukaryotic single-stranded binding protein, is a heterotrimeric complex formed by RPA-1, RPA-2 and RPA-3. RPA is a fundamental player in replication, repair, recombination and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes...
October 16, 2017: Journal of Eukaryotic Microbiology
Chuanhe Yu, Haiyun Gan, Zhiguo Zhang
DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication...
2018: Methods in Molecular Biology
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