Kinetic analysis of nucleotide incorporation by mammalian DNA polymerase delta.
The kinetics of nucleotide incorporation into 24/36-mer primer/template DNA by purified fetal calf thymus DNA polymerase (pol) delta was examined using steady-state and pre-steady-state kinetics. The role of the pol delta accessory protein, proliferating cell nuclear antigen (PCNA), on DNA replication by pol delta was also examined by kinetic analysis. The steady-state parameter k(cat) was similar for pol delta in the presence and absence of PCNA (0.36 and 0.30 min(-1), respectively); however, the K(m) for dNTP was 20-fold higher in the absence of PCNA (0.067 versus 1.2 microm), decreasing the efficiency of nucleotide insertion. Pre-steady-state bursts of nucleotide incorporation were observed for pol delta in the presence and absence of PCNA (rates of polymerization (k(pol)) of 1260 and 400 min(-1), respectively). The reduction in polymerization rate in the absence of PCNA was also accompanied by a 2-fold decrease in burst amplitude. The steady-state exonuclease rate of pol delta was 0.56 min(-1) (no burst, 10(3)-fold lower than the rate of polymerization). The small phosphorothioate effect of 2 for correct nucleotide incorporation into DNA by pol delta.PCNA indicated that the rate-limiting step in the polymerization cycle occurs prior to phosphodiester bond formation. A K(d)(dNTP) value of 0.93 microm for poldelta.dNTP binding was determined by pre-steady-state kinetics. A 5-fold increase in K(d)(DNA) for the pol delta.DNA complex was measured in the absence of PCNA. We conclude that the major replicative mammalian polymerase, pol delta, exhibits kinetic behavior generally similar to that observed for several prokaryotic model polymerases, particularly a rate-limiting step following product formation in the steady state (dissociation of oligonucleotides) and a rate-limiting step (probably conformational change) preceding phosphodiester bond formation. PCNA appears to affect pol delta replication in this model mainly by decreasing the dissociation of the polymerase from the DNA.
Einolf
H J
HJ
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. heidi.einolf@pharma.novartis.com
Guengerich
F P
FP
D000818:Animals; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D004258:DNA Polymerase III / Q000737:chemistry*; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000737:chemistry*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D013950:Thymus Gland / Q000196:embryology
D017931:DNA Primers; D004268:DNA-Binding Proteins; D003845:Deoxycytosine Nucleotides; D009711:Nucleotides; D018809:Proliferating Cell Nuclear Antigen; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D004258:DNA Polymerase III; D005092:Exonucleases