G:T mismatches, the major mispairs generated during DNA metabolism, are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). Mismatch-specific DNA glycosylases must discriminate the mismatches against million-fold excess correct base pairs. MBD4 efficiently removes thymine opposite guanine but not opposite adenine. Previous studies have revealed that the substrate thymine is flipped out and enters the catalytic site of the enzyme, while the estranged guanine is stabilized by Arg468 of MBD4. To gain further insights into the mismatch discrimination mechanism of MBD4, we assessed the glycosylase activity of MBD4 toward various base pairs. In addition, we determined a crystal structure of MBD4 bound to T:O6-methylguanine-containing DNA, which suggests the O6 and N2 of purine and the O4 of pyrimidine are required to be a substrate for MBD4. To understand the role of the Arg468 finger in catalysis, we evaluated the glycosylase activity of MBD4 mutants, which revealed the guanidinium moiety of Arg468 may play an important role in catalysis. D560N/R468K MBD4 bound to T:G mismatched DNA shows that the side chain amine moiety of the Lys stabilizes the flipped-out thymine by a water-mediated phosphate pinching, while the backbone carbonyl oxygen of the Lys engages in hydrogen bonds with N2 of the estranged guanine. Comparison of various DNA glycosylase structures implies the guanidinium and amine moieties of Arg and Lys, respectively, may involve in discriminating between substrate mismatches and nonsubstrate base pairs.
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Research Article|
May 28 2021
Insights into the substrate discrimination mechanisms of methyl-CpG-binding domain 4
Hala Ouzon-Shubeita;
Hala Ouzon-Shubeita
Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing
The Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, U.S.A.
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Lillian F. Schmaltz;
Lillian F. Schmaltz
The Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, U.S.A.
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Seongmin Lee
Conceptualization, Supervision, Funding acquisition, Validation, Writing - review & editing
The Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, U.S.A.
Correspondence: Seongmin Lee (SeongminLee@austin.utexas.edu)
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Publisher: Portland Press Ltd
Received:
January 15 2021
Revision Received:
May 05 2021
Accepted:
May 07 2021
Accepted Manuscript online:
May 07 2021
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2021
Biochem J (2021) 478 (10): 1985–1997.
Article history
Received:
January 15 2021
Revision Received:
May 05 2021
Accepted:
May 07 2021
Accepted Manuscript online:
May 07 2021
Citation
Hala Ouzon-Shubeita, Lillian F. Schmaltz, Seongmin Lee; Insights into the substrate discrimination mechanisms of methyl-CpG-binding domain 4. Biochem J 28 May 2021; 478 (10): 1985–1997. doi: https://doi.org/10.1042/BCJ20210017
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