Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase.
DNA polymerase-primase complex, isolated with an apparently undegraded alpha-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the alpha-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage phi X174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10(-6) and 10 x 10(-6). This value reflects the spontaneous mutation frequency of phi X174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10(-7) (1/2 880,000) for dGMP:Ttemplate mispairs, between 10(-7) and 10(-8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(-8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'----5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)
Bialek
G
G
Abteilung Chemie, Max-Planck-Institut für experimentelle Medizin, Göttingen, FRG.
Nasheuer
H P
HP
Goetz
H
H
Grosse
F
F
D010584:Bacteriophage phi X 174 / Q000235:genetics / Q000378:metabolism; D001482:Base Composition; D004247:DNA / Q000096:biosynthesis*; D019915:DNA Primase; D004260:DNA Repair; D004261:DNA Replication; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000378:metabolism; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*
D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V