DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli

Abstract

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0. 6) than in a DeltapolB (approximately 0.1) strain. Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.

FIG. 1

The NER/DNA Pol II pathway for the repair of a nitrogen mustard interstrand cross-link. Repair of the top strand is proposed to involve NER (step 1), followed by DNA Pol II filling of the gap, including bypass of the lesion and ligation (steps 2 and 3) and finally NER of the resulting monoadduct in the bottom strand (step 4).

FIG. 2

Survival curves for various strains of E. coli treated with nitrogen mustard (see Materials and Methods). Strains: FC40 (wild type [○]), PFB60 (ΔpolB [●]), FC348 (ΔrecA [□]), B98 (ΔpolB ΔrecA [■]), and PFB50 (uvrA6 [▵]). Panel a shows all of the data, whereas panel b shows the data for the latter three strains with an expanded abscissa. Strain FC348 is strain FC40 with a ΔrecA allele. Strain B98 is strain FC40 with both ΔpolB and ΔrecA alleles. Strain PFB50 is strain FC40 with a uvrA6 allele. Strains are described in Table 1.