Name: application of stopped-flow kinetics methods to investigate the mechanism of action of a dna repair protein
Uploaded: 2010-03-31T17:00:00
Description: Watch this Scientific Journal Video about Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein at JoVE.com

यह काम एक NSF कैरियर अवार्ड (MMH), बैरी एम. Goldwater छात्रवृत्ति (एफएनबी) और एक ASBMB अंडर ग्रेजुएट रिसर्च अवार्ड (CWD) द्वारा समर्थित किया गया था. PBP की अभिव्यक्ति के लिए क्लोन कृपया डॉ. मार्टिन वेब (MRC, ब्रिटेन) द्वारा प्रदान की गई थी.

Msh2 - Msh6 डीएनए बाध्यकारी कैनेटीक्स ए मापन 1. Msh2 Msh6 डीएनए बाध्यकारी कैनेटीक्स प्रयोग के लिए नमूना तैयार प्रतिदीप्ति आधारित एक बंद कर दिया प्रवाह पर गतिज डीएनए बाध्यकारी प्रयोग के लिए अभि...

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct,+real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase.">Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.</a> Biochemistry. 33 (27), 8262-8271 (1994).</li><li>Brune, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+inorganic+phosphate+interaction+with+phosphate+binding+protein+from+Escherichia+coli.">Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.</a> Biochemistry. 37 (29), 10370-10380 (1998).</li><li>Antony, E., Hingorani, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+ATP+binding+and+hydrolysis+activity+of+the+Thermus+aquaticus+MutS+dimer+is+key+to+modulation+of+its+interactions+with+mismatched+DNA.">Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA.</a> Biochemistry. 43 (41), 13115-13128 (2004).</li><li>Gradia, S., Acharya, S., Fishel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+mismatch+recognition+complex+hMSH2-hMSH6+functions+as+a+novel+molecular+switch.">The human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch.</a> Cell. 91 (7), 995-1005 (1997).</li><li>Mazur, D. J., Mendillo, M. L., Kolodner, R. 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उदाहरण के एक डीएनए बेमेल बाध्यकारी प्रोटीन यहाँ वर्णित की शक्ति और जैविक अणुओं के तंत्र के अध्ययन के लिए क्षणिक गतिज तरीकों की उपयोगिता दिखाता है. एकल कारोबार समय के पैमाने पर रूका प्रवाह माप एक बेमेल ...

<table border="1"><tr><td> डीएनए नाम </td><td> अनुक्रम </td></tr><tr><td> 37 जी </td><td> टीसीसी 5&#39;-ATT टीटीसी AGC आगा जैसे जी टीए सीसीए टीएसी TGA टीटीसी एसीए टी -3 &#39; </td></tr><tr><td> 37 टी (2 - एपी) </td><td> TGA 5&#39;-ATG एटीसी AGT ATG GTA टी पी टी एटीसी TGC TGA AGG एएए टी -3 &#39; </td></tr><tr><td> 37 टी </td><td> 5&#39;-ATG TGA एटीसी AGT ATG GTA एक टी एटीसी TGC TGA AGG एएए टी -3 टी &#39; </td></tr></table>

application of stopped-flow kinetics methods to investigate the mechanism of action of a dna repair protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Watch this Scientific Journal Video about Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein at JoVE.com

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Msh2 - Msh6 डीएनए में प्रतिकृति त्रुटियों की मरम्मत की शुरुआत के लिए जिम्मेदार है. यहाँ हम समझने की दिशा में एक क्षणिक कैनेटीक्स दृष्टिकोण है कैसे काम करता है इस महत्वपूर्ण प्रोटीन मौजूद है. रिपोर्ट मिलकर डीएनए बाध्यकारी और ATPase कैनेटीक्स अंतर्निहित डीएनए की मरम्मत में Msh2 Msh6 कार्रवाई के तंत्र को मापने के लिए बंद कर दिया प्रवाह के प्रयोगों को दिखाता है.

रूका प्रवाह तरीके कैनेटीक्स के आवेदन के लिए एक डीएनए मरम्मत प्रोटीन की कार्रवाई के तंत्र की जांच

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

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