Fidelity Mechanisms of DNA Polymerase Alpha
Abstract
We have examined the mechanism by which DNA polymerase alpha discriminates between right and wrong dNTPs. With purine dNTPs, the enzyme uses a combination of positive and negative selectivity. The Watson-Crick hydrogen bonding groups at N-1 and C-6 enhance correct incorporation, whereas the electron density at N-1 and N-3 prevent misincorporation. For correct purine dNTPs, the electron density of N-3 is not essential. The exocyclic N-2 amino group of dGTP is not essential for incorporation, but does specifically prevent formation of G:A mispairs. With pyrimidine dNTPs, O-2 appears critical for incorporation. Thus, minor groove electron density appears critical for correct incorporation of pyrimidine dNTPs, but not purine dNTPs. Importantly, the roles of the functional groups vary substantially depending upon whether the modified base is in the template or incoming dNTP, indicating that pol alpha likely "reads" the template prior to selecting the correct incoming dNTP. Single turnover rapid quench and "trapping" approaches were developed for analyzing dNTP polymerization by pol ?. Initial experiments have indicated both that pol ? binds DNA with moderate affinity and that dNTP polymerization is only moderately fast.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 23, 2008
- Accession Number
- ADA499543
Entities
People
- Robert D. Kuchta
Organizations
- University of Colorado Boulder