DNA synthesis by CHO cell extracts on fork-like DNA templates containing the major cisplatin adduct requires a ligation step

In this work we have examined the role of DNA ligation in the in vitro repl ication catalyzed by CHO crude extracts on fork-like oligonucleotide substr ates containing a unique d(GpG) intrastrand cross-link produced by the anti tumor drug cisplatin. We show here that this reaction involves a ligation s tep, which necessitates excision of the flap strand of the forked substrate . By constructing substrates in which the unannealed tail could not be degr aded by a 5' exonuclease, we obtained evidence suggesting that this type of activity participates in the removal of the flap strand. Furthermore, we f ound that the ligation event played a predominant role in the synthesis of fully replicated products from both intact and platinated templates. Finall y, we investigated whether translesion synthesis of the cisplatin lesion co uld occur concomitantly to ligation by monitoring the incorporation of labe led precursors downstream of the adduct. Our results are compatible with th e possibility that some translesion syntheses of the Pt-d(GpG) adduct by th e extracts also contributed to the generation of full length molecules. (C) 2000 Societe francaise de biochimie et biologie moleculaire/Editions scien tifiques et medicales Elsevier SAS.