Substrate Recognition and Catalysis by the Holliday Junction Resolving Enzyme Hje

CL Middleton, Joanne Louise Parker, Derek John Richard, Malcolm Frederick White, CS Bond

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)

Abstract

Two archaeal Holliday junction resolving enzymes, Holliday junction (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns-they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.

Original languageEnglish
Pages (from-to)5442-5451
JournalNucleic Acids Research
Volume32
Issue number18
DOIs
Publication statusPublished - 2004

Keywords

  • T7 ENDONUCLEASE-I
  • X-RAY-DIFFRACTION
  • PROTEIN DATA-BANK
  • SULFOLOBUS-SOLFATARICUS
  • CRYSTAL-STRUCTURE
  • ESCHERICHIA-COLI
  • ACTIVE-SITE
  • PYROCOCCUS-FURIOSUS
  • DNA RECOGNITION
  • BINDING

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