HU PROTEIN OF ESCHERICHIA-COLI BINDS SPECIFICALLY TO DNA THAT CONTAINS SINGLE-STRAND BREAKS OR GAPS

In this study, we have identified a protein in Escherichia coli that s pecifically binds to double-stranded DNA containing a single-stranded gap of one nucleotide. The gap-DNA binding (GDB) protein was purified to apparent homogeneity, The analysis of the amino-terminal sequencing of the GDB protein shows two closely related sequences we identify as the alpha and beta subunits of the HU protein, Furthermore, the GDB p rotein is not detected in the crude extract of an E. coli double mutan t strain hupA hupB that has no functional HU protein. These results le d us to identify the GDB protein as the HU protein. HU binds strongly to double stranded 30-mer oligonucleotides containing a nick or a sing le-stranded gap of one or two nucleotides, Apparent dissociation const ants were measured for these various DNA duplexes using a gel retardat ion assay, The K-D(app) values were 8 nM for the 30-mer duplex that co ntains a nick and 4 and 2 nM for those that contain a 1- or a 2-nucleo tide gap, respectively, The affinity of HU for these ligands is at lea st 100-fold higher than for the same 30-mer DNA duplex without nick or gap. Other single-stranded breaks or gaps, which are intermediate pro ducts in the repair of abasic sites after incision by the Fpg, Nth, or Nfo proteins, are also preferentially bound by the HU protein, Due to specific: binding to DNA strand breaks, HU may play a role in replica tion, recombination, and repair.