Cruciform DNA binding protein in HeLa cell extracts.
We have analyzed by band-shift assays HeLa cell protein-DNA interactions on a stable cruciform DNA molecule. The stable cruciform was formed by heteroduplexing the HindIII-SphI fragment of SV40 virus DNA that contains the origin of replication with a derivative mutant containing a heterologous substitution at the central inverted repeat. We have identified a novel binding activity in HeLa cell extracts with specificity for the cruciform-containing DNA and no apparent sequence specificity. The activity is protein-dependent, void of detectable nuclease activity, and distinct from that reported for HMG1. A cruciform binding protein (CBP) with an apparent molecular weight of 66 kDa was enriched from HeLa cell extracts. In addition to the CBP, we have detected sequence-specific binding activities to sites proximal to the cruciform. Binding to one such site is increased in the cruciform-containing heteroduplex DNA by comparison to its linear homoduplex counterpart, suggesting transmission of structural effects by the stem-loops to their local environment.
Pearson
C E
CE
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Ruiz
M T
MT
Price
G B
GB
Zannis-Hadjopoulos
M
M
D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; D019286:Endopeptidase K; D006367:HeLa Cells / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000494:pharmacology; D013539:Simian virus 40 / Q000235:genetics; D013329:Structure-Activity Relationship
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D015247:Deoxyribonuclease HindIII; C046184:endodeoxyribonuclease PaeI; D015252:Deoxyribonucleases, Type II Site-Specific; D012259:Ribonuclease, Pancreatic; D012697:Serine Endopeptidases; D019286:Endopeptidase K