Unwinding of unnatural substrates by a DNA helicase

Helicases separate double-stranded DNA into single-stranded DNA intermediat es that are required during replication and recombination. These enzymes ar e believed to transduce free energy available from ATPase activity to unwin d the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates h as not been well-defined. Most helicases require a single-stranded DNA over hang adjacent to duplex DNA in order to initiate unwinding. The strand cont aining the overhang is referred to as the loading strand whereas the comple mentary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA) . PNA is capable of forming duplex structures with DNA according to Watson- Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone i n place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melti ng temperatures than their DNA-DNA counterparts. Dda helicase, from bacteri ophage T4, was able to unwind the DNA-PNA substrates at similar rates as DN A-DNA substrates. The results indicate that the rate-limiting step for unwi nding is relatively insensitive to the chemical nature of the displaced str and and the thermal stability of oligonucleotide substrates.