Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I

Abstract

A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I (DNA pol I). Escherichia coli DNA pol I bypassed the lesion efficiently, but partial inhibition was observed for T4 DNA polymerase. The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center. These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase-DNA-dNTP complex. When primer elongation is not possible, proofreading becomes the favored reaction. Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion. The replication block was overcome by higher dNTP concentrations. Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks.

Figure 1

Replication of an ethyl phosphotriester lesion by wild-type T4 DNA polymerase. Control (C) or ethyl phosphotriester (PTE) DNA substrates (Table 1) were replicated by the wild-type T4 DNA polymerase in reactions with either 5 or 100 µM dNTPs. The primer was labeled with ³²P as described in the Material and Methods. Primer extension reactions were run from 20 s to 10 min as indicated. Denaturing gel electrophoresis was used to separate the products. If the ethyl phosphotriester blocked replication, a build-up of +7 product was produced.

Figure 2

Replication of an ethyl phosphotriester lesion by exonuclease-deficient T4 DNA polymerase. The reaction conditions were the same as described for the wild-type T4 DNA polymerase (Fig. 1).

Figure 3

Replication of an ethyl phosphotriester lesion by the T4 I417V–DNA polymerase. The reaction conditions were the same as described for the wild-type T4 DNA polymerase (Fig. 1).

Figure 4

Replication of an ethyl phosphotriester lesion by E.coli DNA pol I. The reaction conditions were the same as described for the wild-type T4 DNA polymerase (Fig. 1).

Figure 5

TLR of an ethyl phosphotriester lesion by T4 DNA polymerase. The region of the DNA substrate containing the ethyl phosphotriester is illustrated; the full DNA substrate is described in Table 1. The green circles indicate the phosphodiester linkages. The ethyl phosphotriester adduct is illustrated by an Et, which may be in one of the two positions indicated. (Note that only a single ethyl phosphotriester, the Sp or Rp isomer, was present in individual DNA substrates.) Either idling opposite template T₇ or primer elongation can occur at the lesion. Additional details are supplied in the text.

Figure 6

RB69 DNA polymerase contacts with oxygen atoms in phosphodiester linkages in the primer terminal region of the template strand. The primer terminal region of the template strand for the ternary DNA polymerase–DNA–dNTP structure reported by Franklin et al. (23) for the RB69 DNA polymerase is shown in the figure. The template strand is forced into an abrupt turn, which is between template bases A and C. Protein contacts are made with the Rp oxygen in the turn region and with the Sp oxygen in the phosphodiester linkage between template bases G and A. Additional details are supplied in the text.