EVIDENCE FOR A RECOMBINATION-INDEPENDENT PATHWAY FOR THE REPAIR OF DNA INTERSTRAND CROSS-LINKS BASED ON A SITE-SPECIFIC STUDY WITH NITROGEN-MUSTARD

DNA-DNA interstrand cross-links are thought to be important for the cy totoxicity of many chemotherapeutic agents. To study this more definit ively, adduct site-specific methods are used to construct a plasmid wi th a single nitrogen mustard interstrand cross-link (inter-HN2-pTZSV28 ). Replication efficiency (RE = [colonies from (inter-HN2-pTZSV28)/(co ntrol with no cross-link)]) is similar to 0.3 following transformation into Escherichia coli, implying that the cross-link is repaired. The commonly accepted pathway for cross-link repair, which involves both n ucleotide excision repair (NER) and recombination, is ruled out since RE is similar to 0.3 in a drecA strain. Non-RecA-directed recombinatio n such as copy-choice is also unlikely. However, NER is involved since RE was similar to 0.02 in strains deficient in NER. Base excision rep air is not important since RE is similar to 0.3 in strains deficient i n 3-methyladenine DNA glycosylases I and II, FAPY DNA glycosylase, bot h known apurinic/apyrimidinic endonucleases, or DNA deoxyribophosphodi esterase. Another hypothetical repair pathway hinging on a 5' --> 3' e xonuclease activity is unlikely since RE is similar to 0.3 in cells de ficient in either the 5' --> 3' exonuclease activities of DNA polymera se I, exonuclease VII, or RecJ. Thus, aside from NER, it is unclear wh at else participates in this recombination-independent repair pathway, although a pathway involing NER followed by replicative bypass of the lesion is the current working hypothesis. Psoralen interstrand cross- links appear not to be repairable by this second pathway, which may ha ve implications for the relative cytotoxicity of interstrand cross-lin ks from different agents.