Bypass of N²-ethylguanine by human DNA polymerase κ.
The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N(2)-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3' termini opposite N(2)-ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N(2)-ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn(2+) compared to Mg(2+). In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N(2)-ethyl-Gua bypass is Mg(2+)-dependent. Strikingly, Mn(2+) activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N(2)-ethyl-Gua and Gua compared to that detected upon Mg(2+) activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high K(m) values for dCTP insertion opposite N(2)-ethyl-Gua and Gua with Mn(2+) activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn(2+) compared to Mg(2+) indicating that DNA pol κ preferentially utilizes Mg(2+) activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N(2)-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn(2+) suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis.
Department of Biochemistry, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA.
Matthew G
MG
Pence
Patrick
P
Blans
Charles N
CN
Zink
James C
JC
Fishbein
Fred W
FW
Perrino
D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D003845:Deoxycytosine Nucleotides; D006147:Guanine; D008274:Magnesium; D008345:Manganese; D011993:Recombinant Fusion Proteins; C073641:N(2)-ethylguanine; C024107:2'-deoxycytidine 5'-triphosphate; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases