Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver.
Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.
Shevelev
I V
IV
Laboratory of DNA Biosynthesis, Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute of the Russia Academy of Sciences, Gatchina, Leningrad, Russia.
Belyakova
N V
NV
Kravetskaya
T P
TP
Krutyakov
V M
VM
D000818:Animals; D010584:Bacteriophage phi X 174 / Q000201:enzymology; D002417:Cattle; D002843:Chromatin / Q000737:chemistry; D002850:Chromatography, Gel; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D004256:DNA Polymerase I / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009152:Mutagenicity Tests; D051381:Rats; D012044:Regression Analysis; D013950:Thymus Gland / Q000737:chemistry / Q000201:enzymology; D014158:Transcription, Genetic / Q000187:drug effects
D002843:Chromatin; D003848:Deoxyguanine Nucleotides; C007257:2'-deoxyguanosine 5'-phosphate; C029603:deoxyguanosine triphosphate; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V