Extremophiles

, Volume 11, Issue 5, pp 675–684

A single amino acid substitution in the DNA-binding domain of Aeropyrum pernix DNA ligase impairs its interaction with proliferating cell nuclear antigen

  • Shinichi Kiyonari
  • Toru Kamigochi
  • Yoshizumi Ishino
Original Paper

DOI: 10.1007/s00792-007-0083-0

Cite this article as:
Kiyonari, S., Kamigochi, T. & Ishino, Y. Extremophiles (2007) 11: 675. doi:10.1007/s00792-007-0083-0

Abstract

Proliferating cell nuclear antigen (PCNA) is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair. Previously, it was reported that a crenarchaeal PCNA formed a heterotrimeric structure, and that each PCNA subunit has distinct binding specificity to PCNA-binding proteins. Here we describe the PCNA-binding properties of a DNA ligase from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. Based on our findings on the Pyrococcus furiosus DNA ligase–PCNA interaction, we predicted that the aromatic residue, Phe132, in the DNA-binding domain of A. pernix DNA ligase (ApeLig) would play a critical role in binding to A. pernix PCNA (ApePCNA). Surface plasmon resonance analyses revealed that the ApeLig F132A mutant does not interact with an immobilized subunit of ApePCNA. Furthermore, we could not detect any stimulation of the ligation activity of the ApeLig F132A protein by ApePCNA in vitro. These results indicated that the phenylalanine, which is located in our predicted PCNA-binding region in ApeLig, has a critical role for the physical and functional interaction with ApePCNA.

Keywords

Archaea Aeropyrum pernix K1 DNA ligase Proliferating cell nuclear antigen PIP box DNA replication 

Abbreviations

DBD

DNA-binding domain

DTT

Dithiothreitol

FEN-1

Flap endonuclease 1

IDCL

Interdomain connecting loop

K-Glu

Potassium glutamate

PCNA

Proliferating cell nuclear antigen

PIP box

PCNA interaction protein box

SPR

Surface plasmon resonance

Copyright information

© Springer 2007

Authors and Affiliations

  • Shinichi Kiyonari
    • 1
    • 2
  • Toru Kamigochi
    • 1
    • 2
  • Yoshizumi Ishino
    • 1
    • 2
  1. 1.Department of Genetic Resources Technology, Faculty of AgricultureKyushu UniversityFukuokaJapan
  2. 2.BIRD-Japan Science and Technology AgencyFukuokaJapan

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