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                            Library & Multimedia
 Prediction of Active Site and Distal Residues in E.coli DNA Polymerase III alpha Polymerase Activity
NCJ Number
              253357
Journal

*Biochemistry* Volume: 57 Issue: 7 Dated: 2018 Pages: 1063-1072
Author(s)
          Ramya Parasuram;
                        Timothy A. Coulther;
                        Judith M. Hollander;
                        Elise Keston-Smith;
                        Mary J. Ondrechen;
                        Penny J. Beuning
Date Published

2018
Length
              10 pages
Annotation
              The goal of this research was to determine the residues that contribute to the activity of the polymerase subunit of E.coli DNA Pol III.
Abstract
              The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10 different subunits that coordinates simultaneous replication on the leading and lagging strands. The 1160-residue Pol III alpha subunit is responsible for the polymerase activity and copies DNA accurately, making one error per 105 nucleotide incorporations. In the current study, homology modeling and the computational methods of THEMATICS and POOL were used to predict functionally important amino acid residues through their computed chemical properties. Site-directed mutagenesis and biochemical assays were used to validate these predictions. Primer extension, steady-state single-nucleotide incorporation kinetics, and thermal denaturation assays were performed to understand the contribution of these residues to the function of the polymerase. This work shows that the top 15 residues predicted by POOL, a set that includes the three previously known catalytic aspartate residues, seven remote residues, plus five previously unexplored first-layer residues, are important for function. Six previously unidentified residues, R362, D405, K553, Y686, E688, and H760, are each essential to Pol III activity; three additional residues, Y340, R390, and K758, play important roles in activity. (publisher abstract modified)
      
  
          Additional Details
Grant Number(s)
        2015-R2-CX-0011


  
  
        
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  Publication Type
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  Award
        Engineering a high-fidelity, damage-tolerant polymerase for forensic analysis of damaged DNA samples
    
  
  
Date Published: January 1, 2018
    
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