The ligation reaction was performed as described previously [32] . Briefly, a 36 bp DNA duplex harboring a centrally placed nick was used as a substrate. The 18-mer constituting the 59phosphate-terminated strand 59-d(ATTCCGATAGTGACTA-CA)-39 was 59-radiolabeled and gel purified as described before. This labeled 18-mer was then annealed to a complementary 36mer in the presence of a 39-OH 18-mer strand 59-d(CAT-ATCCGTGTCGCCCTT)-39 [38, 39] . Ligation reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 10 mM MgCl 2 , 1 mM ATP, nicked duplex substrate and the Chlorella virus DNA ligase were incubated at 22uC for 15 min. The reactions were stopped by the addition of 1 ml 0.5 M EDTA and 5 ml formamide. The samples were heated at 95uC for 5 min and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea. The extent of ligation was determined by scanning the gel with a PhosphorImager (Amersham Biosciences).