Human DNA polymerase kappa forms nonproductive complexes with matched primer termini but not with mismatched primer termini.
Human DNA polymerase kappa (pol kappa) is a member of the Y family of DNA polymerases that function in translesion synthesis. It synthesizes DNA with moderate fidelity and does not efficiently incorporate nucleotides opposite DNA lesions. Pol kappa has the unusual ability to efficiently extend from mismatched primer termini, and it extends readily from nucleotides inserted by other DNA polymerases opposite a variety of DNA lesions. All of this has suggested that pol kappa functions during the extension step of translesion synthesis. Here, we have carried out pre-steady-state kinetic studies of pol kappa using DNA with matched and mismatched primer termini. Interestingly, we find that mismatches present only a modest kinetic barrier to nucleotide incorporation by pol kappa. Moreover, and quite surprisingly, active-site titrations revealed that the concentration of active pol kappa is very low with matched DNA, and from DNA trapping experiments we determined that this was due to the formation of nonproductive protein.DNA complexes. In marked contrast, we found that the concentration of active pol kappa was six-fold greater with mismatched DNA than with matched DNA. Thus, pol kappa forms nonproductive complexes with matched but not with mismatched DNA. From these observations, we conclude that pol kappa has evolved to specifically function on DNA substrates with aberrant primer-terminal base pairs, such as the ones it would encounter during the extension step of translesion synthesis.
Carlson
Karissa D
KD
Department of Biochemistry, University of Iowa College of Medicine, 51 Newton Road, Iowa City, IA 52242-1109, USA.
Johnson
Robert E
RE
Prakash
Louise
L
Prakash
Satya
S
Washington
M Todd
MT
D020137:Base Pair Mismatch / Q000235:genetics*; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013379:Substrate Specificity; D017186:Titrimetry; D000099270:Y-Family DNA Polymerases
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases