Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV

Abstract

Repair of interstrand DNA cross-links (ICLs) in Escherichia coli can occur through a combination of nucleotide excision repair (NER) and homologous recombination. However, an alternative mechanism has been proposed in which repair is initiated by NER followed by translesion DNA synthesis (TLS) and completed through another round of NER. Using site-specifically modified oligodeoxynucleotides that serve as a model for potential repair intermediates following incision by E. coli NER proteins, the ability of E. coli DNA polymerases (pol) II and IV to catalyze TLS past N(2)-N(2)-guanine ICLs was determined. No biochemical evidence was found suggesting that pol II could bypass these lesions. In contrast, pol IV could catalyze TLS when the nucleotides that are 5' to the cross-link were removed. The efficiency of TLS was further increased when the nucleotides 3' to the cross-linked site were also removed. The correct nucleotide, C, was preferentially incorporated opposite the lesion. When E. coli cells were transformed with a vector carrying a site-specific N(2)-N(2)-guanine ICL, the transformation efficiency of a pol II-deficient strain was indistinguishable from that of the wild type. However, the ability to replicate the modified vector DNA was nearly abolished in a pol IV-deficient strain. These data strongly suggest that pol IV is responsible for TLS past N(2)-N(2)-guanine ICLs.

FIGURE 1.

DNA substrates. A, schematic representation of DNA substrates: ICL1, ICL2, ICL3, and ICL4 containing an interstrand cross-link and a non-damaged substrate (ND). Arrows indicate the direction of DNA synthesis. The site of the N²-N²-guanine cross-link is underlined. dd, dideoxynucleotide; gl, glycerol. B, structure of N²-N²-guanine cross-link. C, primer sequences used in the in vitro reactions. D, schematic representation of an N²-N²-guanine cross-link-containing 36-mer oligodeoxynucleotide that was used for generating the pMS2-ICL vector.

FIGURE 2.

A and B, primer extension by pol II and pol IV, respectively. Time course primer extension experiments were conducted using non-damaged and ICL DNA substrates (ICL1, ICL2, ICL3, and ICL4). A –10 primer (10 bases upstream of the adduct site) was annealed to the DNA template and incubated with pol II or pol IV (0.3 nm) for the indicated times. C, primer extension activity by pol IV is dose-dependent. The experiment was conducted using non-damaged and ICL1–4 templates primed with the –10 primer with increasing concentrations of pol IV at 37 °C for 30 min. M indicates the oligodeoxynucleotide size marker (Fisher).

FIGURE 3.

Accuracy of pol IV-catalyzed nucleotide incorporation opposite the N²-N²-guanine cross-link. A single nucleotide incorporation assay was conducted in the presence of pol IV (0.3 nm) and individual nucleotides (25 μm) using the –1 primer annealed to non-damaged or ICL4 substrate (5 nm). These reactions were carried out for 30 min at 37 °C. M indicates the oligodeoxynucleotide size marker (Fisher).

FIGURE 4.

Relative colony-forming ability of E. coli strains following transformation with an ICL-containing plasmid. For each strain, the percentage of pMS2-ICL transformants was calculated relative to pBR322 transformants. The apparent transformation efficiency with the reference plasmid, pBR322, was comparable for all the strains tested.