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1.
EMBO J ; 35(18): 2045-59, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27481934

ABSTRACT

8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxo-dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post-replicative 8-oxo-dG:dA mispairs are removed through DNA polymerase lambda (Pol λ)-dependent MUTYH-initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8-oxo-dG bypass by Pol λ. We demonstrate that Pol λ has a flexible active site that can tolerate 8-oxo-dG in either the anti- or syn-conformation. Importantly, we show that discrimination against the pro-mutagenic syn-conformation occurs at the extension step and identify the residue responsible for this selectivity. This residue acts as a kinetic switch, shunting repair toward long-patch BER upon correct dCMP incorporation, thus enhancing repair efficiency. Moreover, this switch also provides a potential mechanism to increase repair fidelity of MUTYH-initiated BER.


Subject(s)
Base Pair Mismatch , DNA Polymerase beta/chemistry , DNA Polymerase beta/metabolism , DNA Repair , Deoxyguanosine/analogs & derivatives , 8-Hydroxy-2'-Deoxyguanosine , Catalytic Domain , Crystallography, X-Ray , Deoxyguanosine/metabolism , Humans , Kinetics , Protein Conformation
2.
Nucleic Acids Res ; 41(16): 7947-59, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23804760

ABSTRACT

Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabetes. Here, we present the first structural characterization of the mammalian TFB1 factor. We have solved two X-ray crystallographic structures of TFB1M with (2.1 Å) and without (2.0 Å) its cofactor S-adenosyl-L-methionine. These structures reveal that TFB1M shares a conserved methyltransferase core with other KsgA/Dim1 methyltransferases and shed light on the structural basis of S-adenosyl-L-methionine binding and methyltransferase activity. Together with mutagenesis studies, these data suggest a model for substrate binding and provide insight into the mechanism of methyl transfer, clarifying the role of this factor in an essential process for mitochondrial function.


Subject(s)
DNA-Binding Proteins/chemistry , Methyltransferases/chemistry , Mitochondrial Proteins/chemistry , S-Adenosylmethionine/chemistry , Transcription Factors/chemistry , Animals , DNA-Binding Proteins/metabolism , Humans , Ligands , Methyltransferases/metabolism , Mice , Mitochondrial Proteins/metabolism , Models, Molecular , Protein Conformation , S-Adenosylmethionine/metabolism , Transcription Factors/metabolism
3.
Structure ; 20(11): 1940-7, 2012 Nov 07.
Article in English | MEDLINE | ID: mdl-23022348

ABSTRACT

MTERF4 is the first MTERF family member shown to bind RNA and plays an essential role as a regulator of ribosomal biogenesis in mammalian mitochondria. It forms a complex with the rRNA methyltransferase NSUN4 and recruits it to the large ribosomal subunit. In this article, we characterize the interaction between both proteins, demonstrate that MTERF4 strongly stimulates the specificity of NSUN4 during in vitro methylation experiments, and present the 2.0 Å resolution crystal structure of the MTERF4:NSUN4 protein complex, lacking 48 residues of the MTERF4 C-terminal acidic tail, bound to S-adenosyl-L-methionine, thus revealing the nature of the interaction between both proteins and the structural conservation of the most divergent of the human MTERF family members. Moreover, the structure suggests a model for RNA binding by the MTERF4:NSUN4 complex, providing insight into the mechanism by which an MTERF family member facilitates rRNA methylation.


Subject(s)
Methyltransferases/chemistry , RNA, Ribosomal/metabolism , Transcription Factors/chemistry , Amino Acid Sequence , Methyltransferases/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Transcription Factors/metabolism
4.
Curr Med Chem ; 19(25): 4348-58, 2012.
Article in English | MEDLINE | ID: mdl-22830350

ABSTRACT

Matrix metalloproteinases (MMPs) are essential for the degradation and turnover of components of the extracellular matrix (ECM) and, when pathologically elevated, mediate connective tissue loss (including bone destruction) in various inflammatory and other diseases. Tetracyclines (TCs) are known inhibitors of mammalian-derived MMPs, and non-antibiotic formulations of Doxycycline are FDA-approved to treat periodontitis and the chronic inflammatory skin disease, rosacea. Because the C-11/ C-12 diketonic moiety of the tetracyclines is primarily responsible, through zinc-binding, for MMP inhibition, we have uniquely modified curcumin as a "core" molecule, since it contains a similar enolic system and is known to have beneficial effects in diseases where connective-tissue loss occurs. Specifically we have developed new congeners which exhibit improved zinc-binding and solubility, and potent reduction of excessive MMP levels and activity. We now describe a series of curcuminoid bi- and tri-carbonylmethanes in which all of these properties are substantially improved. An N-phenylaminocarbonyl derivative of bis-demethoxycurcumin (CMC2.24) was selected as the "lead" substance because it showed superior potency in vitro (i.e., the lowest IC(50)) against a series of neutral proteases (MMPs) associated with tissue erosion. Moreover, CMC2.24 administered to diabetic rats orally (30mg/kg), reduced the secretion of pathologically-excessive levels of MMP-9 to normal in cultured peritoneal macrophages with no evidence of toxicity. Thus, this (and other similar novel) compound(s) may be useful in various diseases of connective-tissue loss.


Subject(s)
Curcumin/analogs & derivatives , Curcumin/chemistry , Curcumin/therapeutic use , Diabetes Mellitus, Type 1/drug therapy , Matrix Metalloproteinase Inhibitors/chemistry , Matrix Metalloproteinase Inhibitors/therapeutic use , Matrix Metalloproteinases/metabolism , Animals , Cell Line , Cells, Cultured , Curcumin/pharmacology , Diabetes Mellitus, Type 1/enzymology , Drug Design , Humans , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/enzymology , Male , Matrix Metalloproteinase Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley
5.
Mitochondrion ; 12(5): 492-9, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22784680

ABSTRACT

The MTERF protein family comprises members from Metazoans and plants. All the Metazoan MTERF proteins characterized to date, including the mitochondrial transcription termination factors, play a key role in mitochondrial gene expression. In this study we report the characterization of Drosophila MTERF5 (D-MTERF5), a mitochondrial protein existing only in insects, probably originated from a duplication event of the transcription termination factor DmTTF. D-MTERF5 knock-down in D.Mel-2 cells alters transcript levels with an opposite pattern to that produced by DmTTF knock-down. D-MTERF5 is able to interact with mtDNA at the same sites contacted by DmTTF, but only in the presence of the termination factor. We propose that the two proteins participate in the transcription termination process, with D-MTERF5 engaged in relieving the block exerted by DmTTF. This hypothesis is supported also by D-MTERF5 homology modeling, which suggests that this protein contains protein-protein interaction domains. Co-regulation by DREF (DNA Replication-related Element binding Factor) of D-MTERF5 and DmTTF implies that expression of the two factors needs to be co-ordinated to ensure fine modulation of Drosophila mitochondrial transcription.


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Gene Expression Regulation , Mitochondria/genetics , Mitochondrial Proteins/metabolism , Transcription, Genetic , Animals , Cell Line , DNA-Binding Proteins/genetics , Drosophila , Drosophila Proteins/genetics , Gene Knockdown Techniques , Mitochondrial Proteins/genetics , Protein Interaction Domains and Motifs , Sequence Homology, Amino Acid
6.
Cell ; 141(6): 982-93, 2010 Jun 11.
Article in English | MEDLINE | ID: mdl-20550934

ABSTRACT

Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that the mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.


Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , DNA, Mitochondrial/metabolism , Terminator Regions, Genetic , Transcription, Genetic , Amino Acid Sequence , Basic-Leucine Zipper Transcription Factors/chemistry , Basic-Leucine Zipper Transcription Factors/genetics , DNA, Mitochondrial/chemistry , Humans , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Mitochondrial Proteins , Models, Molecular , Nucleotides/metabolism , Point Mutation , RNA, Transfer, Leu/genetics
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