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PDBsum entry 3khb
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Oxidoreductase
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PDB id
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3khb
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References listed in PDB file
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Key reference
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Title
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Structural and mutational analysis of escherichia coli alkb provides insight into substrate specificity and DNA damage searching.
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Authors
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P.J.Holland,
T.Hollis.
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Ref.
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Plos One, 2010,
5,
e8680.
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PubMed id
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Abstract
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BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position
of purines and N3 position of pyrimidines are primarily repaired by the
2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs
1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also
repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less
efficient rate. How the AlkB enzyme is able to locate and identify methylated
bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We
determined the crystal structures of the E. coli AlkB protein holoenzyme and the
AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed
mutagenesis of amino acids in and around the active site, and tested the effects
of these mutations on the ability of the protein to bind both damaged and
undamaged DNA, as well as catalyze repair of a methylated substrate.
CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex
with our unliganded holoenzyme reveals conformational changes of residues within
the active site that are important for binding damaged bases. Site-directed
mutagenesis of these residues reveals novel insight into their roles in DNA
damage recognition and repair. Our data support a model that the AlkB protein
utilizes at least two distinct conformations in searching and binding methylated
bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to
functionally separate these modes through mutagenesis of residues that affect
one or the other binding state. Finally, our mutagenesis experiments show that
amino acid D135 of AlkB participates in both substrate specificity and catalysis.
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