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Divergent β-hairpins determine double-strand versus single-strand substrate recognition of human AlkB-homologues 2 and 3
- Source :
- Nucleic Acids Research
- Publication Year :
- 2010
- Publisher :
- Oxford University Press (OUP), 2010.
-
Abstract
- Human AlkB homologues ABH2 and ABH3 repair 1-methyladenine and 3-methylcytosine in DNA/RNA by oxidative demethylation. The enzymes have similar overall folds and active sites, but are functionally divergent. ABH2 efficiently demethylates both single- and double-stranded (ds) DNA, whereas ABH3 has a strong preference for single-stranded DNA and RNA. We find that divergent F1 β-hairpins in proximity of the active sites of ABH2 and ABH3 are central for substrate specificities. Swapping F1 hairpins between the enzymes resulted in hybrid proteins resembling the donor proteins. Surprisingly, mutation of the intercalating residue F102 had little effect on activity, while the double mutant V101A/F102A was catalytically impaired. These residues form part of an important hydrophobic network only present in ABH2. In this functionally important network, F124 stacks with the flipped out base while L157 apparently functions as a buffer stop to position the lesion in the catalytic pocket for repair. F1 in ABH3 contains charged and polar residues preventing use of dsDNA substrate. Thus, E123 in ABH3 corresponds to F102 in ABH2 and the E123F-variant gained capacity to repair dsDNA with no loss in single strand repair capacity. In conclusion, divergent sequences outside of the active site determine substrate specificities of ABH2 and ABH3.
- Subjects :
- Models, Molecular
DNA repair
AlkB
DNA, Single-Stranded
Genome Integrity, Repair and Replication
medicine.disease_cause
Protein Structure, Secondary
Dioxygenases
Substrate Specificity
chemistry.chemical_compound
Catalytic Domain
Genetics
medicine
Humans
chemistry.chemical_classification
Mutation
biology
AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase
Active site
RNA
Substrate (chemistry)
DNA
DNA Repair Enzymes
Enzyme
chemistry
Biochemistry
Mutagenesis, Site-Directed
biology.protein
AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase
Hydrophobic and Hydrophilic Interactions
Subjects
Details
- ISSN :
- 13624962 and 03051048
- Volume :
- 38
- Database :
- OpenAIRE
- Journal :
- Nucleic Acids Research
- Accession number :
- edsair.doi.dedup.....e8184fec59522368adbf3eae452b939c