1xo1
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(New page: 200px<br /><applet load="1xo1" size="450" color="white" frame="true" align="right" spinBox="true" caption="1xo1, resolution 2.5Å" /> '''T5 5'-EXONUCLEASE MUT...)
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Revision as of 04:06, 21 November 2007
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T5 5'-EXONUCLEASE MUTANT K83A
Overview
Efficient cellular DNA replication requires the activity of a 5'-3', exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA, substrates exonucleolytically, and they are structure-specific, endonucleases. The 5'-3' exonucleases are conserved in organisms as, diverse as bacteriophage and mammals. Crystal structures of three, representative enzymes identify two divalent-metal-binding sites typically, separated by 8-10 A. Site-directed mutagenesis was used to investigate the, roles of three lysine residues (K83, K196, and K215) situated near two, metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196, nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the, substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical, analysis demonstrated that K83 is absolutely required for exonucleolytic, activity on single-stranded DNA but is not required for endonucleolytic, cleavage of flap structures. Structural analysis of this mutant by x-ray, crystallography showed no significant perturbations around the, metal-binding sites in the active site. The wild-type protein has, different pH optima for endonuclease and exonuclease activities. Taken, together, these results suggest that different mechanisms for endo- and, exonucleolytic hydrolysis are used by this multifunctional enzyme.
About this Structure
1XO1 is a Single protein structure of sequence from Pseudomonas phage d3112. Active as Exodeoxyribonuclease (lambda-induced), with EC number 3.1.11.3 Full crystallographic information is available from OCA.
Reference
Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage., Garforth SJ, Ceska TA, Suck D, Sayers JR, Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):38-43. PMID:9874768
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