Determining the DNA bending angle induced by non-specific high mobility group-1 (HMG-1) proteins: a novel method.
To study the DNA bending induced by non-sequence-specific HMG-1 domain proteins, we have engineered a fusion protein linking the yeast NHP6A with a sequence-specific DNA binding domain, the DNA binding domain of the Hin recombinase, Hin-DBD. A series of biochemical experiments were carried out to characterize the DNA binding property of this fusion protein. Our data showed that the fusion protein not only specifically recognizes a DNA fragment containing the Hin-DBD binding site, but also binds DNA with a higher affinity in comparison with either domain alone. Both domains of the fusion protein are bound to the DNA in juxtaposition. Permutation assays showed that the fusion protein induced a DNA bending at the site of NHP6A binding by an estimated value of 63 degrees. We believe that this experimental design provides an effective vehicle to determine the DNA bending induced by nonspecific HMG-1 proteins.
Tang
L
L
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Li
J
J
Katz
D S
DS
Feng
J A
JA
D015374:Biosensing Techniques / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024001:HMGN Proteins; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D024001:HMGN Proteins; D006609:High Mobility Group Proteins; C086471:NHP6A protein, S cerevisiae; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; C059997:Hin recombinase