CONTRASTING EFFECTS OF SINGLE-STRANDED-DNA BINDING-PROTEIN ON THE ACTIVITY OF URACIL DNA GLYCOSYLASE FROM ESCHERICHIA-COLI TOWARDS DIFFERENT DNA SUBSTRATES

Excision of uracil from tetraloop hairpins and single stranded ('unstr uctured') oligodeoxyribonucleotides by Escherichia coli uracil DNA gly cosylase has been investigated. We show that, compared with a single s tranded reference substrate, uracil from the first, second, third and the fourth positions of the loops is excised with highly variable effi ciencies of 3.21, 0.37, 5.9 and 66.8%, respectively. More importantly, inclusion of E.coli single stranded DNA binding protein (SSB) in the reactions resulted in similar to 7-140-fold increase in the efficiency of uracil excision from the first, second or the third position in th e loop but showed no significant effect on its excision from the fourt h position. In contrast, the presence of SSB decreased uracil excision from the single stranded ('unstructured') substrates similar to 2-3-f old. The kinetic studies show that the increased efficiency of uracil release from the first, second and the third positions of the tetraloo ps is due to a combination of both the improved substrate binding and a large increase in the catalytic rates. On the other hand, the decrea sed efficiency of uracil release from the single stranded substrates ( 'unstructured') is mostly due to the lowering of the catalytic rates. Chemical probing with KMnO4 showed that the presence of SSB resulted i n the reduction of cleavage of the nucleotides in the vicinity of dUMP residue in single stranded substrates but their increased susceptibil ity in the hairpin substrates. We discuss these results to propose tha t excision of uracil from DNA-SSB complexes by uracil DNA glycosylase involves base flipping. The use of SSB in the various applications of uracil DNA glycosylase is also discussed.