High affinity interactions of nucleolin with G-G-paired rDNA.
Nucleolin is a very abundant eukaryotic protein that localizes to the nucleolus, where the rDNA undergoes transcription, replication, and recombination and where rRNA processing occurs. The top (non-template) strand of the rDNA is very guanine-rich and has considerable potential to form structures stabilized by G-G pairing. We have assayed binding of endogenous and recombinant nucleolin to synthetic oligonucleotides in which G-rich regions have formed intermolecular G-G pairs to produce either two-stranded G2 or four-stranded G4 DNA. We report that nucleolin binds G-G-paired DNA with very high affinity; the dissociation constant for interaction with G4 DNA is KD = 1 nM. Two separate domains of nucleolin can interact with G-G-paired DNA, the four RNA binding domains and the C-terminal Arg-Gly-Gly repeats. Both domains bind G4 DNA with high specificity and recognize G4 DNA structure independent of sequence context. The high affinity of the nucleolin/G4 DNA interaction identifies G-G-paired structures as natural binding targets of nucleolin in the nucleolus. The ability of two independent domains of nucleolin to bind G-G-paired structures suggests that nucleolin can function as an architectural factor in rDNA transcription, replication, or recombination.
Hanakahi
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Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510-8024, USA.
Sun
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Maizels
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D000818:Animals; D020029:Base Pairing / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000737:chemistry*; D006801:Humans; D051379:Mice; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D000097796:Nucleolin
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D006147:Guanine; D009687:Nuclear Proteins; D009841:Oligonucleotides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000097796:Nucleolin