Contacts between the 5 ' nuclease of DNA polymerase I and its DNA substrate

The 5 ' nuclease of DNA polymerase I (Pol I) of Escherichia coli is a membe r of an important class of prokaryotic and eukaryotic nucleases, involved i n DNA replication and repair, with specificity for the junction between sin gle-stranded and duplex DNA. We have investigated the interaction of the 5 ' nuclease domain with DNA substrates from the standpoint of both the prote in and the DNA. Phosphate ethylation interference showed that the nuclease binds to the nucleotides immediately surrounding the cleavage site and also contacts the complementary strand one-half turn away, indicating that cont acts are made to one face only of the duplex portion of the DNA substrate. Phosphodiester contacts were investigated further using DNA substrates carr ying unique methylphosphonate substitutions, together with mutations in the 5 ' nuclease. These experiments suggested that two highly conserved basic residues, Lys(78) and Arg(81), are close to the phosphodiester immediately 5 ' to the cleavage site, while a third highly conserved residue, Arg(20), may interact with the phosphodiester 3 ' to the cleavage site. Our results provide strong support for a DNA binding model proposed for the related exo nuclease from bacteriophage T5, in which the conserved basic residues menti oned above define the two ends of a helical arch that forms part of the sin gle-stranded DNA-binding region. The nine highly conserved carboxylates in the active site region appear to play a relatively minor role in substrate binding, although they are crucial for catalysis. In addition to binding th e DNA backbone around the cleavage point, the 5 ' nuclease also has a bindi ng site for one or two frayed bases at the 3 ' end of an upstream primer st rand. In agreement with work in related systems, 5 ' nuclease cleavage is b locked by duplex DNA in the 5 ' tag, but the enzyme is quite tolerant of ab asic DNA or polarity reversal within the 5 ' tail.