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

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemothe rapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escher ichia coli, and its replication efficiency (RE = [number of transformants f rom inter-HN2-pTZSV28]/[number of transformants from control]) was determin ed to be similar to 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 t he cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (R E) is higher in a polB(+) (similar to 0.6) than in a Delta polB (similar to 0.1) strain. Complementation with a polB(+)-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate exper iments, E.coli was treated with HN2, and the relative sensitivity to killin g was found to be as follows: wild type < polB < recA < polB recA similar t o uvrA. Because cells deficient in either recombination (recA) or DNA polym erase II (polB) are hypersensitive to nitrogen mustard killing, E. coli app ears to have two pathways for cross-link repair: an NER/recombination pathw ay (which is possible when the cross-links are formed in tells where recomb ination can occur because there are multiple copies of the genome) and an N ER/DNA polymerase II pathway. Furthermore, these results show that some cro ss-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 a coli. It remains to be determined why this new pathway prefers DNA polymer ase II and why there are two pathways to repair cross-links.