The Experts below are selected from a list of 8817 Experts worldwide ranked by ideXlab platform
Matthew R Redinbo - One of the best experts on this subject based on the ideXlab platform.
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tyrosine partners coordinate DNA Nicking by the salmonella typhimurium plasmid pcu1 relaxase enzyme
FEBS Letters, 2011Co-Authors: Rebekah P Nash, Franklin Niblock, Matthew R RedinboAbstract:Conjugative plasmid transfer results in the spread of antibiotic resistance genes and virulence factors between bacterial cells. Plasmid transfer is dependent upon the DNA Nicking activity of a plasmid-encoded relaxase enzyme. Tyrosine residues within the relaxase cleave the DNA plasmid nic site in a highly sequence-specific manner. The conjugative resistance plasmid pCU1 encodes a relaxase with four tyrosine residues surrounding its active site (Y18,19,26,27). We use activity assays to demonstrate that the pCU1 relaxase preferentially uses Y26 or a combination of Y18 + 19 to Nick DNA at wild type levels, and that an adjacent aspartic acid deprotonates these tyrosines to activate them for attack. Our findings illustrate the unique modifications that the pCU1 relaxase has introduced into the traditional relaxase-mediated DNA Nicking mechanism.
Xiaozhuo Chen - One of the best experts on this subject based on the ideXlab platform.
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seryl histidine as an alternative DNA Nicking agent in Nick translation yields superior DNA probes and hybridizations
Bioorganic & Medicinal Chemistry, 2002Co-Authors: Yunsheng Li, Scott Hatfield, Jing Li, Mark C Mcmills, Yufen Zhao, Xiaozhuo ChenAbstract:Abstract Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNAse I to generate Nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3′ hydroxyl and 5′ phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNAse I as an alternative DNA Nicking agent. In this study we demonstrate that like DNAse I, Ser-His randomly Nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through Nick translation using Ser-His as the DNA Nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNAse I as the Nicking agent. Furthermore, the degree of Nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His Nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA Nicking agent in Nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated Nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may Nick DNA.
Melike Caglayan - One of the best experts on this subject based on the ideXlab platform.
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the scaffold protein xrcc1 stabilizes the formation of polβ gap DNA and ligase iiiα Nick DNA complexes in base excision repair
Journal of Biological Chemistry, 2021Co-Authors: Qun Tang, Melike CaglayanAbstract:The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent Nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of Nicked repair products to the final ligation step. Moreover, the mutagenic ligation of Nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/Nick DNA catalytic ternary complexes. Real-time monitoring of protein–protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for Nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.
Rebekah P Nash - One of the best experts on this subject based on the ideXlab platform.
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tyrosine partners coordinate DNA Nicking by the salmonella typhimurium plasmid pcu1 relaxase enzyme
FEBS Letters, 2011Co-Authors: Rebekah P Nash, Franklin Niblock, Matthew R RedinboAbstract:Conjugative plasmid transfer results in the spread of antibiotic resistance genes and virulence factors between bacterial cells. Plasmid transfer is dependent upon the DNA Nicking activity of a plasmid-encoded relaxase enzyme. Tyrosine residues within the relaxase cleave the DNA plasmid nic site in a highly sequence-specific manner. The conjugative resistance plasmid pCU1 encodes a relaxase with four tyrosine residues surrounding its active site (Y18,19,26,27). We use activity assays to demonstrate that the pCU1 relaxase preferentially uses Y26 or a combination of Y18 + 19 to Nick DNA at wild type levels, and that an adjacent aspartic acid deprotonates these tyrosines to activate them for attack. Our findings illustrate the unique modifications that the pCU1 relaxase has introduced into the traditional relaxase-mediated DNA Nicking mechanism.
David R Edgell - One of the best experts on this subject based on the ideXlab platform.
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the monomeric giy yig homing endonuclease i bmoi uses a molecular anchor and a flexible tether to sequentially Nick DNA
Nucleic Acids Research, 2013Co-Authors: Benjamin P Kleinstiver, Jason M Wolfs, David R EdgellAbstract:The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially Nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.