A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase β Cancer-Associated Variant.
K289M is a variant of DNA polymerase β (pol β) that has previously been identified in colorectal cancer. The expression of this variant leads to a 16-fold increase in mutation frequency at a specific site in vivo and a reduction in fidelity in vitro in a sequence context-specific manner. Previous work shows that this reduction in fidelity results from a decreased level of discrimination against incorrect nucleotide incorporation at the level of polymerization. To probe the transition state of the K289M mutator variant of pol β, single-turnover kinetic experiments were performed using β,γ-CXY dGTP analogues with a wide range of leaving group monoacid dissociation constants (pKa4), including a corresponding set of novel β,γ-CXY dCTP analogues. Surprisingly, we found that the values of the log of the catalytic rate constant (kpol) for correct insertion by K289M, in contrast to those of wild-type pol β, do not decrease with increased leaving group pKa4for analogues with pKa4values of <11. This suggests that one of the relative rate constants differs for the K289M reaction in comparison to that of the wild type (WT). However, a plot of log(kpol) values for incorrect insertion by K289M versus pKa4reveals a linear correlation with a negative slope, in this respect resembling kpolvalues for misincorporation by the WT enzyme. We also show that some of these analogues improve the fidelity of K289M. Taken together, our data show that Lys289 critically influences the catalytic pathway of pol β.
Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Khadijeh S
0000-0002-0219-6807
KS
Alnajjar
Department of Chemistry, University of Southern California , Los Angeles, California 90089, United States.
Beatriz
B
Garcia-Barboza
Department of Chemistry, University of Southern California , Los Angeles, California 90089, United States.
Amirsoheil
A
Negahbani
Department of Chemistry, University of Southern California , Los Angeles, California 90089, United States.
Maryam
M
Nakhjiri
Department of Chemistry, University of Southern California , Los Angeles, California 90089, United States.
Boris
B
Kashemirov
Department of Chemistry, University of Southern California , Los Angeles, California 90089, United States.
Charles
C
McKenna
Department of Chemistry, University of Southern California , Los Angeles, California 90089, United States.
Myron F
MF
Goodman
Department of Therapeutic Radiology and Department of Genetics, Yale University School of Medicine , New Haven, Connecticut 06520, United States.
Joann B
JB
Sweasy
D015179:Colorectal Neoplasms / Q000201:enzymology*; D019951:DNA Polymerase beta / Q000378:metabolism*; D007700:Kinetics; D058105:Polymerization; D013379:Substrate Specificity
D019951:DNA Polymerase beta