Nucleotide excision by E. coli DNA polymerase I in proofreading and non-proofreading modes.
Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms. When it binds to a template, the proofreading activity is usually switched off. As the enzyme progresses along the template, it becomes more and more competent for excision. This phenomenon introduces a link between fidelity and processivity. Processivity is best studied when the chain-length distributions of synthesized polymers are stationary. Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme. When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed. It seems that the polymerase starts by hydrolyzing the primer down to a length of 7-10 nucleotides and only then starts to add nucleotides. It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading. The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3'----5' exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity.
Lecomte
P J
PJ
Institut Jacques Monod, Paris, France.
Ninio
J
J
D004256:DNA Polymerase I / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D013698:Templates, Genetic
D004269:DNA, Bacterial; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; C027903:oligo (dT); D011061:Poly A; C015465:poly(dA); D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V