Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje.
Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (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.
Middleton
Claire L
CL
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
Parker
Joanne L
JL
Richard
Derek J
DJ
White
Malcolm F
MF
Bond
Charles S
CS
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D045566:DNA, Cruciform / Q000737:chemistry / Q000378:metabolism*; D019143:Evolution, Molecular; D045565:Holliday Junction Resolvases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology; D014764:Viral Proteins / Q000235:genetics
D019843:Archaeal Proteins; D045566:DNA, Cruciform; D013431:Sulfates; D014764:Viral Proteins; D012694:Serine; D045565:Holliday Junction Resolvases