Sequence-dependent contribution of distal binding domains to CAP protein-DNA binding affinity

Abstract Abstract 4193 The MLL gene encodes a multi-domain protein that is involved in the maintenance of Hox gene expression during development and hematopoiesis, and was first identified through its involvement in chromosome translocations that cause leukemia. The CXXC domain of MLL, which is retained in leukemic MLL fusion proteins, is a cysteine rich DNA binding domain, with specificity for binding nonmethylated CpG-containing DNA, and is essential for MLL fusion proteins' oncogenic properties. We performed domain swap experiments in which CXXC domains from other proteins were swapped in to replace MLL's CXXC domain in the context of an oncogenic MLL fusion. CXXC domains from DNA methyltransferase 1 (DNMT1), CpG binding protein (CGBP), and methyl-CpG binding domain protein 1 (MBD1), as well as a methyl binding domain (MBD) from MBD1 were swapped into the MLL-AF9 fusion. These particular domains were chosen because their described CpG DNA binding capacity is either similar or different from that described for MLL. In vitro colony assays on isolated murine bone marrow progenitor cells infected with domain swapped or wild type MLL-AF9 fusion genes were performed in order to determine whether CpG binding domains from other proteins would affect the ability of MLL-AF9 to give an enhanced proliferative capacity to bone marrow progenitor cells. In vivo murine studies determined whether the different CpG binding domains alter the ability of MLL fusion proteins to cause leukemia. We predicted that the different CpG binding domains would change the strength or specificity of MLL binding to DNA, which would affect the ability of MLL-AF9 to cause leukemia. The results of both in vitro replating assays and in vivo leukemogenesis experiments have shown significant differences between the ability of various CpG DNA binding domains to function in the context of an MLL-AF9 fusion protein. MLL-AF9 containing the DNMT1 CXXC domain shows robust in vitro colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all show reduced colony forming ability and leukemogenicity in vivo. In vitro DNA binding experiments are currently being performed to directly measure and compare the DNA binding affinity of the CXXC domain from MLL to the other domain swap proteins. Preliminary data suggests that MLL CXXC has a stronger DNA binding affinity to non-methylated DNA compared to the other CXXC domains. Furthermore, the DNMT and CGBP CXXC domains both show lower affinity DNA binding compared to MLL CXXC, but they have different effects in MLL-AF9. This suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to an MLL fusion protein's leukemogenic properties. Disclosures: No relevant conflicts of interest to declare.

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The Molecular Mechanisms of Allosteric Mutations Impairing MepR Repressor Function in Multidrug-Resistant Strains of Staphylococcus aureus

mBio ◽

10.1128/mbio.00528-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 15

Author(s):

Ivan Birukou ◽

Nam K. Tonthat ◽

Susan M. Seo ◽

Bryan D. Schindler ◽

Glenn W. Kaatz ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Dna Binding ◽

Family Member ◽

Binding Affinity ◽

Multidrug Resistant ◽

Content Type ◽

Linker Region ◽

Dna Binding Domains ◽

Binding Domains ◽

Dna Binding Affinity

ABSTRACTOverexpression of theStaphylococcus aureusmultidrug efflux pump MepA confers resistance to a wide variety of antimicrobials.mepAexpression is controlled by MarR family member MepR, which repressesmepAand autorepresses its own production. Mutations inmepRare a primary cause ofmepAoverexpression in clinical isolates of multidrug-resistantS. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasingmepRoperator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistantStaphylococcus aureus.IMPORTANCEStaphylococcus aureusis a major health threat to immunocompromised patients.S. aureusmultidrug-resistant variants that overexpress the multidrug efflux pumpmepAemerge frequently due to point mutations in MarR family member MepR, themepAtranscription repressor. Significantly, the majority of MepR mutations identified in theseS. aureusclinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistantStaphylococcus aureus.

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Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells

Methods in Molecular Biology - Functional Genomics ◽

10.1007/978-1-4939-7231-9_27 ◽

2017 ◽

pp. 361-375 ◽

Cited By ~ 2

Author(s):

Mir A. Hossain ◽

Isaac J. Knudson ◽

Shaleen Thakur ◽

Yong Shen ◽

Jared R. Stees ◽

...

Keyword(s):

Dna Binding ◽

Binding Affinity ◽

Zinc Finger ◽

Protein Delivery ◽

Mammalian Cells ◽

Dna Binding Domains ◽

Binding Domains ◽

Dna Binding Affinity

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Faculty Opinions recommendation of Reduction in DNA-binding affinity of Cys2His2 zinc finger proteins by linker phosphorylation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019055.215578 ◽

2004 ◽

Author(s):

Philip Dawson

Keyword(s):

Dna Binding ◽

Binding Affinity ◽

Zinc Finger ◽

Zinc Finger Proteins ◽

Dna Binding Affinity

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Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Scientific Reports ◽

10.1038/s41598-021-94528-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Krystyna Ślaska-Kiss ◽

Nikolett Zsibrita ◽

Mihály Koncz ◽

Pál Albert ◽

Ákos Csábrádi ◽

...

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Binding Affinity ◽

Dna Methyltransferase ◽

Restriction Enzymes ◽

Dna Binding Protein ◽

E Coli ◽

Dna Binding Affinity ◽

Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

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