High mobility group proteins 1 and 2 can function as DNA-binding regulatory components for DNA-dependent protein kinase in vitro

Yoshiko Yumoto, Hitoshi Shirakawa, Michiteru Yoshida, Akira Suwa, Fumiaki Watanabe, Hirobumi Teraoka

Research output: Contribution to journalArticlepeer-review

28 Citations (Scopus)

Abstract

The DNA-dependent protein kinase (DNA-PK) holoenzyme consists of a 470-kDa catalytic subunit (DNA-PKcs), a DNA-binding regulatory component known as Ku protein, and double-stranded DNA (dsDNA) with ends. We previously reported that the activity of DNA-PK in vitro is stimulated by non-histone chromosomal high mobility group proteins (HMG) 1 and 2 comprising two similar repeats, termed domains A and B, and an acidic C-terminal. Here we demonstrate that in vitro HMG1 and 2 can completely replace Ku protein as the DNA-binding regulatory component of DNA-PK. DNA-PKcs and Ku protein were separately purified from Raji nuclear extracts, and reconstituted into the DNA-PK holoenzyme in the presence of dsDNA, DNA-PKcs alone catalyzed DNA-dependent phosphorylation at a very low but significant level, and HMG1 and 2 markedly stimulated the phosphorylation of α-casein and a specific peptide substrate in a DNA-dependent manner. The HMG2-domains (A + B) polypeptide devoid of the C-terminal acidic region was more effective for DNA-PKcs stimulation than the full-length HMG2, and HMG2-domain A and -domain B polypeptides. Anti(Ku protein) antibodies inhibited the DNA-dependent phosphorylation activity of the DNA-PKcs:Ku protein complex, but not that of DNA-PKcs alone or when it was complexed with HMG1 or 2. These results demonstrate that HMG1 and 2 can function as the DNA-binding regulatory component for DNA-PKcs in vitro, and imply that a conformational change of dsDNA, which is elicited by regulatory components, is important for the stimulation of DNA-PK activity of DNA-PKcs.

Original languageEnglish
Pages (from-to)519-527
Number of pages9
JournalJournal of biochemistry
Volume124
Issue number3
DOIs
Publication statusPublished - 1998 Sep
Externally publishedYes

Keywords

  • DNA-dependent protein kinase
  • Double-stranded DNA
  • HMG1
  • HMG2
  • Ku protein

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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