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 specifically 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 protein is not detected in the crude extract of an E. coli double mutant strain hupA hupB that has no functional HU protein. These results led us to identify the GDB protein as the HU protein. HU binds strongly to double-stranded 30-mer oligonucleotides containing a nick or a single-stranded gap of one or two nucleotides. Apparent dissociation constants were measured for these various DNA duplexes using a gel retardation assay. The KD(app) values were 8 nM for the 30-mer duplex that contains a nick and 4 and 2 nM for those that contain a 1-or a 2-nucleotide gap, respectively. The affinity of HU for these ligands is at least 100-fold higher than for the same 30-mer DNA duplex without nick or gap. Other single-stranded breaks or gaps, which are intermediate products 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 replication, recombination, and repair.
Castaing
B
B
Groupe Réparation des Lésions Radio- et Chimio-Induites, URA 147, CNRS, Institut Gustave Roussy, Villejuif, France.
Zelwer
C
C
Laval
J
J
Boiteux
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008024:Ligands; C040330:histone-like protein HU, bacteria