Abstract
The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3'-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates. DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus. The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were either relatively G + C- or A + T-rich. The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3'-terminal nucleotides were unpaired. Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction. The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase. These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.
Highlights
Duces the exonuclease activity to barely detectable levels [5,6,7]
The kinetic traces for the hydrolysis reactions with the 2AP-T and 2AP-A DNA substrates appeared to be biphasic
The 2AP-T and 2AP-C DNA substrates were selected for further study because of the respective biphasic and monophasic kinetics observed for the hydrolysis reactions for the two substrates, and because of the relevance of proofreading 2AP-T and 2AP-C primer termini to understanding how 2AP promotes base substitution mutagenesis
Summary
Duces the exonuclease activity to barely detectable levels [5,6,7]. Another reaction step in proofreading that may be affected by mutation is the translocation of the primer end of the DNA between the spatially distinct polymerase and exonuclease active centers. The wild type level of 3Ј 3 5Ј-exonuclease activity was detected with single-stranded DNA substrates which indicates that the ability to hydrolyze a phosphodiester bond is not affected in the mutant enzyme; significantly reduced activity was observed with double-stranded DNAs [9]. These results suggest that some aspect in the process of converting duplex DNA with a mispaired primer terminus, the natural substrate for proofreading, into the more strand-separated DNA required for 3Ј 3 5Ј-exonuclease activity is defective in the mutant. In order to investigate the strand separation step, we designed a series of DNA substrates with an increasing number of mispairs at the primer terminus in order to simulate varying degrees of strand separation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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.
