Abstract
BackgroundIn 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 FindingsWe 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/SignificanceA 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.
Highlights
DNA in cells is constantly exposed to alkylating agents from both the external environment as well as internal cellular metabolic processes
The DNA direct repair protein, Escherichia coli AlkB, repairs DNA alkylation damage that primarily occurs from SN2 alkylating agents [4,5]
The structure of the ssDNA complex was determined using a D135 to Ala (D135A) mutation of AlkB because our mutagenesis data revealed that this residue is responsible for the specificity of 1-meA and 3-meC lesions
Summary
DNA in cells is constantly exposed to alkylating agents from both the external environment as well as internal cellular metabolic processes. These alkylating compounds react primarily with oxygen and nitrogen atoms on DNA bases [1,2] generating lesions that if left unrepaired can be cytotoxic and mutagenic [3]. The AlkB enzyme recognizes and repairs other alkylated DNA lesions such as 3-methylthymine (3-meT) [10], 1-methylguanine (1-meG) [11,12], and 1,N6-ethenoadenine (eA) [13], albeit with lower efficiency. AlkB repairs 1-methyladenine (1meA) and 3-methylcytosine (3-meC) lesions, but it 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
