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Phosphorylation weakens DNA binding by peptides containing multiple "SPKK" sequences

G R Green1, H J Lee, D L Poccia

  • 1Department of Biology, Amherst College, Massachusetts 01002.

The Journal of Biological Chemistry
|May 25, 1993
PubMed
Summary
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Dephosphorylation of sea urchin H1 histone N-terminal regions increases their DNA affinity. This finding is crucial for understanding histone regulation during sperm development.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Spermatogenesis Research

Background:

  • Sea urchin testis-specific H1 and H2B histones (Sp H1 and Sp H2B) feature N-terminal regions rich in "SPKK" motifs.
  • These regions undergo reversible phosphorylation, influencing their interaction with DNA.

Purpose of the Study:

  • To investigate the impact of N-terminal region phosphorylation on the DNA binding affinity of sea urchin H1 histone.
  • To elucidate the role of dephosphorylation in enhancing histone-DNA interactions during spermatogenesis.

Main Methods:

  • Purification of Sp H1 and its phosphorylated form (pSp H1) using hydroxylapatite chromatography.
  • Generation and purification of N-terminal peptides (NP and pNP) via protease digestion and DNA-cellulose chromatography.
  • Comparative analysis of DNA affinities using DNA-cellulose binding, DNA precipitation, DNA thermal denaturation protection, and inhibition of Hoechst 33258 binding assays.

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Main Results:

  • Dephosphorylated N-terminal peptides (NP) exhibited higher affinity for DNA-cellulose compared to phosphorylated forms (pNP).
  • NP effectively precipitated DNA across a range of NaCl concentrations, unlike pNPs.
  • NP formed more stable DNA-peptide complexes and was a significantly more potent inhibitor of Hoechst 33258 DNA binding than pNP.

Conclusions:

  • Dephosphorylation of the Sp H1 N-terminal region significantly increases its basicity and consequently enhances its affinity for DNA.
  • These findings highlight the importance of post-translational modifications in regulating histone-DNA interactions during male gamete formation.