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Title: Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function
Abstract Modified DNA bases functionally distinguish the taxonomic forms of life—5-methylcytosine separates prokaryotes from eukaryotes and 5-hydroxymethylcytosine (5hmC) invertebrates from vertebrates. We demonstrate here that mouse endonuclease G (mEndoG) shows specificity for both 5hmC and Holliday junctions. The enzyme has higher affinity (>50-fold) for junctions over duplex DNAs. A 5hmC-modification shifts the position of the cut site and increases the rate of DNA cleavage in modified versus unmodified junctions. The crystal structure of mEndoG shows that a cysteine (Cys69) is positioned to recognize 5hmC through a thiol-hydroxyl hydrogen bond. Although this Cys is conserved from worms to mammals, a two amino acid deletion in the vertebrate relative to the invertebrate sequence unwinds an α-helix, placing the thiol of Cys69 into the mEndoG active site. Mutations of Cys69 with alanine or serine show 5hmC-specificity that mirrors the hydrogen bonding potential of the side chain (C–H < S–H < O–H). A second orthogonal DNA binding site identified in the mEndoG structure accommodates a second arm of a junction. Thus, the specificity of mEndoG for 5hmC and junctions derives from structural adaptations that distinguish the vertebrate from the invertebrate enzyme, thereby thereby supporting a role for 5hmC in recombination processes.  more » « less
Award ID(s):
1905328
PAR ID:
10171235
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
Nucleic Acids Research
Volume:
48
Issue:
7
ISSN:
0305-1048
Page Range / eLocation ID:
3962 to 3974
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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