A single-stranded DNA binding protein from mouse tumor cells specifically recognizes the C-rich strand of the (AGG:CCT)n repeats that can alter DNA conformation.
A protein that binds to a synthetic oligonucleotide of (CCT)12 has been purified from Ehrlich ascites tumor cells by a (CCT)12 affinity chromatography. The protein (p70) has an apparent molecular mass of 70 kDa, as assayed by Southwestern analysis. A competition experiment revealed that p70 binds to (CCT)12, (CCCT)8 and (CCTCCCT)6, but not to (CTT)12, (CT)16 and (CCTGCCT)6, suggesting that p70 has a sequence-specificity. The complementary (AGG)12 and the double stranded DNA did not show the binding. It is also confirmed by S1 nuclease analysis that the (AGG:CCT)12 duplex takes a single-stranded conformation in the absence of the protein. This raises a possibility that the duplex forms two single-stranded loops in chromosomes, the C-rich strand being bound to p70. Structural analysis of the resulting (AGG)12 strand by non-denaturing polyacrylamide gel electrophoresis demonstrated the presence of slower and faster migrated conformers in a neutral pH buffer containing 50 mM NaCl at 5 degrees C. The ratio was dependent on the DNA concentration. Both conformers disappeared in the absence of NaCl. This suggests that (AGG)12 can form intra- and inter-molecular complexes by non-Watson-Crick, guanine:guanine base-pairing. The possible biological function of the (AGG:CCT)n duplex and the p70 is discussed.
Muraiso
T
T
First Department of Biochemistry, Niigata University School of Medicine, Japan.
Nomoto
S
S
Yamazaki
H
H
Mishima
Y
Y
Kominami
R
R
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001667:Binding, Competitive; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity; D013737:Testis / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes