Abstract
T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction. We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Delta5) to grow on E. coli having reduced thioredoxin levels. One triple mutant (K300E,K302E,K304E) could not support the growth of T7Delta5 even in wild type cells. This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin. The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex. The altered polymerase also has a lower binding affinity for double-stranded DNA.
Highlights
Protein-protein interactions are essential for the coordination of the multiple reactions that occur at a replication fork
As described in the Introduction, earlier studies have implicated a 76-amino acid sequence in T7 gene 5 protein as the thioredoxin binding domain [23, 24].2. This sequence, unique to T7 DNA polymerase, is inserted into the thumb region of the polymerase domain based on the homology of T7 DNA polymerase to E. coli DNA polymerase I [25]
The ability to polymerize thousands of nucleotides during each binding cycle, is an important property for DNA polymerases involved in the replication of a chromosome
Summary
Protein-protein interactions are essential for the coordination of the multiple reactions that occur at a replication fork. Gene 5 protein physically interacts with the hexameric gene 4 protein of the phage, a protein that provides both helicase and primase activity at the replication fork to coordinate both leading and lagging strand synthesis (2, 4 – 6) Both the gene 5 and gene 4 proteins in turn interact with the T7 gene 2.5 protein, a single-stranded DNA (ssDNA)1-binding protein that stimulates both polymerase and primase activities [4, 7, 8]. The enhanced activity on dsDNA is most likely due to a higher affinity of the polymerase-thioredoxin complex to the 3Ј-termini, resulting in increased processivity of hydrolysis [14] Studies on both E. coli thioredoxin and T7 gene 5 protein have provided information on the domains in each protein that are important for the interactions of the two proteins. Relatively large segments of the polypeptide chain that constitutes the polymerase active site of E. coli DNA polymerase I can be exchanged with the homologous region of T7 DNA polymerase with retention of polymerase activity [29]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
