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Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence

N Inoue1, P E Pellett

  • 1National Institute of Health, Tokyo, Japan.

Journal of Virology
|August 1, 1995
PubMed
Summary

Researchers investigated the human herpesvirus 6B (HHV-6B) origin-binding protein (OBP) and its DNA binding. They identified a consensus DNA sequence and mapped the binding domain, proposing a two-subdomain model for herpesvirus OBP DNA interaction.

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Human herpesvirus 6B (HHV-6B) shares structural similarities with alphaherpesvirus replication origins.
  • However, HHV-6B and herpes simplex virus type 1 (HSV-1) origin-binding proteins (OBPs) and origins are not interchangeable.

Purpose of the Study:

  • To characterize the interaction between HHV-6B OBP and its DNA origin.
  • To identify the specific DNA sequence recognized by HHV-6B OBP.
  • To map the DNA-binding domain of HHV-6B OBP and compare it with HSV-1 OBP.

Main Methods:

  • Competitive electrophoretic mobility shift assays (EMSAs) with mutated DNA duplexes.
  • Southwestern (protein-DNA) blotting to determine minimal DNA-binding regions.

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Main Results:

  • A consensus DNA binding sequence for HHV-6B OBP (YGWYCWCCY) was deduced, differing from HSV-1 OBP's sequence (YGYTCGCACT).
  • EMSAs mapped the HHV-6B OBP DNA-binding domain to amino acids 482–770, while Southwestern blotting indicated amino acids 657–770 were sufficient.
  • Southwestern blotting identified amino acids 689–851 of HSV-1 OBP as having binding activity, though insufficient in EMSA.

Conclusions:

  • The DNA-binding domains of herpesvirus OBPs likely consist of two subdomains.
  • A conserved subdomain directly contacts DNA, while a less conserved subdomain may regulate binding.
  • This two-subdomain model explains the observed differences in DNA-binding domain mapping between EMSA and Southwestern blotting.