Pre-steady-state kinetic studies of the fidelity of human DNA polymerase mu.
DNA polymerase mu (Polmu), an X-family DNA polymerase, is preferentially expressed in secondary lymphoid tissues with yet unknown physiological functions. In this study, Polmu was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme had a lifetime of <20 min at 37 degrees C, but was stable for over 3 h at 25 degrees C in an optimized reaction buffer. The fidelity of human Polmu was thus determined using pre-steady-state kinetic analysis of the incorporation of single nucleotides into undamaged DNA 21/41-mer substrates at 25 degrees C. Single-turnover saturation kinetics for all 16 possible deoxynucleotide (dNTP) incorporations and for four matched ribonucleotide (rNTP) incorporations were measured under conditions where Polmu was in molar excess over DNA. The polymerization rate (k(p)), binding affinity (K(d)), and substrate specificity (k(p)/K(d)) are 0.006-0.076 s(-1), 0.35-1.8 microM, and (8-64) x10(-3) microM(-1) s(-1), respectively, for matched incoming dNTPs, (2-30) x 10(-5) s(-1), 7.3-135 microM, and (4-61) x 10(-7) microM(-1) s(-1), respectively, for mismatched incoming dNTPs, and (2-73) x 10(-4) s(-1), 45-302 microM, and (7-1300) x 10(-7) microM(-1) s(-1), respectively, for matched incoming rNTPs. The overall fidelity of Polmu was estimated to be in the range of 10(-3)-10(-5) for both dNTP and rNTP incorporations and was sequence-independent. The sugar selectivity, defined as the substrate specificity ratio of a matched dNTP versus a matched rNTP, was measured to be in the range of 492-10959. In addition to a slow and distributive DNA polymerase activity, Polmu was identified to possess a weak strand-displacement activity. The potential biological roles of Polmu are discussed.
Roettger
Michelle P
MP
Department of Biochemistry, Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
Fiala
Kevin A
KA
Sompalli
Susmitha
S
Dong
Yuxia
Y
Suo
Zucai
Z
D001665:Binding Sites; D002021:Buffers; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D002021:Buffers; D017931:DNA Primers; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D012265:Ribonucleotides; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C029603:deoxyguanosine triphosphate; D004247:DNA; C407743:DNA polymerase mu; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate; D014544:Uridine Triphosphate