Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis.

Abstract

The contribution of proofreading to the fidelity ofcatalysis by DNA polymerases has been determined with deoxyribonucleoside [1-thioltriphosphate substrates. These analogues, which contain a sulfur in place of an oxygen on the ar phosphorus, are incorporated into DNA by DNA polymerases at rates similar to those of the corresponding unmodified deoxynucleoside triphosphates. The fidelity of DNA synthesis was measured with *X174 am3 DNA; reversion to wild type occurs most frequently by a single base substitution, a C for a T at position 587. By using avian myeloblastosis virus DNA polymerase and DNA polymerase (3 (enzymes without a proofreading 3'-*5' exonucleolytic activity), substitution ofdeoxycytidine thiotriphosphate in the reaction mixture did not alter fidelity. In contrast, with DNA polymerases from E. coli (DNA polymerase I) and bacteriophage T4 (enzymes containing a proofreading activity), fidelity was markedly reduced with deoxycytidine [1-thio]triphosphate. DNA containing phosphorothioate nucleotides is insensitive to hydrolysis by the exonuclease associated with these prokapyotic DNA polymerases. These combined results indicate that the deoxynucleoside [1-thio]triphosphates have normal base-pairing properties; however, once misinserted by a polymerase, they are not excised by proofreading. Proofreading ofa C:A mismatch at position 587 is thereby found to contribute 20-fold to the fidelity of E. coli DNA polymerase I and a greater amount to the fidelity of bacteriophage T4 DNA polymerase.The ability to correct errors during DNA replication has long been recognized as one important mechanism by which an organism can potentially achieve the highly accurate replication of its genetic information. This concept stems from the observation that prokaryotic DNA polymerases contain an integrally associated 3'--5' exonuclease activity, which can selectively remove mistakes as they occur during polymerization (1). Biochemical support for this concept was obtained by Brutlag and Kornberg (2), who demonstrated that the 3'--5' exonuclease of Escherichia coli DNA polymerase I (Pol I) preferentially removes mismatched bases at primer termini before initiation of polymerization. Support for proofreading in vivo comes from studies with certain mutator (3) and antimutator (4) bacteriophage T4 DNA polymerases. These studies (5-8) correlated spontaneous mutation rates of bacteriophage T4 with the ratio between the polymerization reaction and the excision of a noncomplementary nucleotide at the primer terminus. Also, differences in discrimination between adenosine and its base analogue 2-aminopurine by mutant T4 DNA polymerase (9) can be accounted for by proofreading. Based on the kinetic data with substrate analogues, a number of mathematic models for proofreading have been proposed (10-13).Our continuing interest in determining the relative importance of the several mechanisms available to the cell to achieve high fidelity (14) has led us to assess the contribution of proofreading to accuracy by direct measuremen...

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

“…Most of these mutator polymerases involve mutation in their associated 3'-5' exonuclease activity. In vitro studies ofthe fidelity ofDNA replication suggest that exonuclease activity is important in the faithful replication of DNA (44). With only a few exceptions (35,36), most purified mammalian DNA polymerases lack an associated exonuclease activity.…”

Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.

“…RTs, which lack 3'--5' exonuclease activity and therefore cannot proofread errors made during synthesis (3), have high base substitution error rates as measured on synthetic polynucleotide templates (2,38,39,48,49,51,59). During synthesis on natural DNA templates, avian myeloblastosis virus (AMV) RT has low fidelity for certain base substitution errors at several different nonsense codons in 4)X174 DNA (16,29,33,34,38,44).…”

Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.

“…The method has been FIG. 4 (10,26,30), but the phosphorothioate diester bonds resist the proofreading exonuclease functions of E. coli DNA polymerase I (10). Similarly, the introduction of thio-dAMP near the 3' ends of DNA restriction fragments yields "capped" molecules that resist degradation by exonuclease III (27).…”

“…Thio-derivatized deoxynucleoside triphosphates substituted in the a position have been employed as precursors for the in vitro synthesis ofviral DNAs (9,10), and they permit an analysis of the stereochemistry of the DNA polymerase reaction (11). Some interesting recent applications include the synthesis of a 'S-labeled cDNA of rabbit globin mRNA (12) and the use of dATP[a-35S] for the nick-translation of plasmid DNA (12).…”