Phosphorylation weakens DNA binding by peptides containing multiple "SPKK" sequences.
Sea urchin testis-specific H1 and H2B histones (Sp H1 and Sp H2B) are characterized by reversibly phosphorylated N-terminal regions consisting largely of multiple clustered "SPKK" tetrapeptides (serine-proline adjacent to two basic amino acids). This report presents data showing differences in DNA affinities between peptides containing dephosphorylated and phosphorylated N-terminal regions. Sp H1 and its phosphorylated derivative (pSp H1) were purified by hydroxylapatite chromatography. Peptides containing the N-terminal regions of Sp H1 and pSp H1 (NP and pNP, respectively) were produced by digestion with Staphylococcus aureus protease. NP and two forms of pNP differing in phosphate content were purified by DNA-cellulose chromatography. The DNA affinities of the peptides were compared using several criteria. NP was bound more tightly by DNA-cellulose than pNPs. NP precipitated DNA under a broad range of NaCl concentrations; pNPs did not. Both NP and pNPs protected DNA against thermal denaturation, but NP created a more stable DNA-peptide complex. Thirty to sixty times more pNP than NP was required to obtain equivalent inhibition of Hoechst 33258 binding to DNA. NP did not behave as a competitive inhibitor of DNA binding by Hoechst 33258 binding to DNA. We conclude that during spermatogenesis, dephosphorylation of the Sp H1 N-terminal region increases its basicity and thus its affinity for DNA.
Green
G R
GR
Department of Biology, Amherst College, Massachusetts 01002.
Lee
H J
HJ
Poccia
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D006690:Bisbenzimidazole / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011489:Protein Denaturation; D012617:Sea Urchins / Q000378:metabolism; D013737:Testis / Q000378:metabolism*; D013816:Thermodynamics
D006657:Histones; D010446:Peptide Fragments; D004247:DNA; D006690:Bisbenzimidazole