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Bacteriophage T7 encodes its own DNA polymerase, gp5, which has both polymerase activity and exonuclease activity. Researchers made two sets of mutations in gp5, mutating various aspartate residues to alanine residues. The mutations are D5A/D65A (blue), D475A/D654A (orange) and D5A/D65A/D475A/D654A (green). They then used isotopically labeled dNTPs to quantify the activity of these mutants compared to wild type gp5 (red). The assay results (A and B) are shown below: A dGMP incorp. (pmol) 30 20 (b) D5A/D65A (c) WT DSA/DISA/DH75A/DISHA D475A/DESTA 500 1000 1500 2000 [gp5] (nM) dNMP released/min (pmol) 20 WT DV75A/DESHA DSA/DA/D47A/DUHA OSA/DISA 11 0 100 200 300 400 500 [gp5] (nM) 10 (a) Which data figure is measuring the polymerase activity? Which data figure is measuring the exonuclease activity? To measure the polymerase activity, which phosphate in the dNTPs needed to be isotopically labeled? Based on the data results, which mutations made the polymerase activity dysfunctional? Which mutations made the exonuclease activity dysfunctional?

Bacteriophage T7 encodes its own DNA polymerase, gp5, which has both polymerase activity and exonuclease activity. Researchers made two sets of mutations in gp5, mutating various aspartate residues to alanine residues. The mutations are D5A/D65A (blue), D475A/D654A (orange) and D5A/D65A/D475A/D654A (green). They then used isotopically labeled dNTPs to quantify the activity of these mutants compared to wild type gp5 (red). The assay results (A and B) are shown below: A dGMP incorp. (pmol) 30 20 (b) D5A/D65A (c) WT DSA/DISA/DH75A/DISHA D475A/DESTA 500 1000 1500 2000 [gp5] (nM) dNMP released/min (pmol) 20 WT DV75A/DESHA DSA/DA/D47A/DUHA OSA/DISA 11 0 100 200 300 400 500 [gp5] (nM) 10 (a) Which data figure is measuring the polymerase activity? Which data figure is measuring the exonuclease activity? To measure the polymerase activity, which phosphate in the dNTPs needed to be isotopically labeled? Based on the data results, which mutations made the polymerase activity dysfunctional? Which mutations made the exonuclease activity dysfunctional?

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
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Bacteriophage T7 encodes its own DNA polymerase, gp5, which has both polymerase activity and exonuclease activity. Researchers made two sets of mutations in gp5, mutating various aspartate residues to alanine residues. The mutations are D5A/D65A (blue), D475A/D654A (orange) and D5A/D65A/D475A/D654A (green). They then used isotopically labeled dNTPs to quantify the activity of these mutants compared to wild type gp5 (red). The assay results (A and B) are shown below: A dGMP incorp. (pmol) 30 (b) D5A/D65A (c) WT DSA/DISA/DH75A/D6STA D475A/D651A 500 1000 1500 2000 [gp5] (nm) B dNMP released/min (pmol) 50 40 30 20 WT D475654 DSA/DA/D47A/DUSHA OSA/DISA 4 0 100 200 300 400 500 [gp5) (nm) 10 (a) Which data figure is measuring the polymerase activity? Which data figure is measuring the exonuclease activity? To measure the polymerase activity, which phosphate in the dNTPs needed to be isotopically labeled? Based on the data results, which mutations made the polymerase activity dysfunctional? Which mutations made the exonuclease activity dysfunctional?
Transcribed Image Text:Bacteriophage T7 encodes its own DNA polymerase, gp5, which has both polymerase activity and exonuclease activity. Researchers made two sets of mutations in gp5, mutating various aspartate residues to alanine residues. The mutations are D5A/D65A (blue), D475A/D654A (orange) and D5A/D65A/D475A/D654A (green). They then used isotopically labeled dNTPs to quantify the activity of these mutants compared to wild type gp5 (red). The assay results (A and B) are shown below: A dGMP incorp. (pmol) 30 (b) D5A/D65A (c) WT DSA/DISA/DH75A/D6STA D475A/D651A 500 1000 1500 2000 [gp5] (nm) B dNMP released/min (pmol) 50 40 30 20 WT D475654 DSA/DA/D47A/DUSHA OSA/DISA 4 0 100 200 300 400 500 [gp5) (nm) 10 (a) Which data figure is measuring the polymerase activity? Which data figure is measuring the exonuclease activity? To measure the polymerase activity, which phosphate in the dNTPs needed to be isotopically labeled? Based on the data results, which mutations made the polymerase activity dysfunctional? Which mutations made the exonuclease activity dysfunctional?
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