COURS BIOCHIMIE EL5BCHAM BIOCHIMIE STRUCTURALE. Pages·· MB· Physicochimie de Macromolécules Biochimie Structurale – LISM. Cahier d’Exercices en Biochimie / PCEM1. Protéine / 2 Enzymologie. .. Quelle caractéristique structurale de ces anticorps est ainsi mise en évidence?. Many translated example sentences containing “biochimie structurale” – English- French 3 A- Première partie: biochimie a- biochimie structurale b- enzymologie c- biochimie . offering a course in biochemistry but without a course [ ].
|Published (Last):||17 May 2012|
|PDF File Size:||19.74 Mb|
|ePub File Size:||8.61 Mb|
|Price:||Free* [*Free Regsitration Required]|
Active DNA demethylation in mammals occurs via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine 5hmC by the ten-eleven translocation family of proteins TETs. Here, we report detailed biochemical and structural characterization of human MBD4 which contains mismatch-specific TDG activity. Here, we also report six crystal structures of human MBD4 cat: These structures reveal that MBD4 cat uses a base flipping mechanism to specifically recognize thymine and 5hmU.
Post-replicative methylation of cytosine at the 5-position 5mC in DNA provides molecular basis of the epigenetic regulation of gene expression 1. MBD4 is a nuclear protein and co-localizes to heterochromatin sites in mouse cells in DNA methylation-dependent manner 78. DNA demethylation occurs either in a passive way via inhibition of de novo methylation after DNA replication, or by an active process, such as direct enzymatic removal of 5mC residues from DNA.
Recent advances in understanding the mechanisms of active DNA demethylation in mammals have identified the ten-eleven translocation family of proteins TETs as 5-methylcytosine 5mC hydroxymethylases. TETs convert 5mC to 5-hydroxymethylcytosine 5hmC and then further oxidize it biochimid 5-formylcytosine 5fC and 5-carboxylcytosine 5caCboth in vitro and in vivo 18— These findings suggest a new unexpected role of the mismatch-specific thymine—uracil DNA glycosylases in the control of epigenetic information via removal of oxidation and deamination products of 5mC.
All oligodeoxyribonucleotides containing modified residues and their complementary oligonucleotides were purchased from Eurogentec Seraing, Belgium including the following: Unless otherwise stated, mer oligonucleotides where target residues are located in XpG context were biocimie in the DNA repair assays. In addition, the purity and integrity of the oligonucleotide preparations were verified by denaturing polyacrylamide gel electrophoresis PAGE.
Collection of the purified DNA glycosylases was from the laboratory stock The concentration of purified proteins was determined by the method of Bradford.
Crystallization conditions are summarized in Table 1. Data collection and processing statistics are given in Table 1. Structure determination of all crystals was performed by molecular replacement with PHASER 28 using first the coordinates of the free-liganded structure PDB code 3IHO for our free-liganded structure at a better resolution and next our model for the DNA—protein structures.
The refined models include residues from to Refinement details of the six structures are courd in Table 1. The resulting samples were desalted by enzymooogie spin-down columns filled with Sephadex G25 Amersham Biosciences equilibrated in 7.
To measure the kinetic parameters of DNA glycosylases-catalysed excision of modified bases, reactions were performed under single turnover conditions. The data were fitted by non-linear regression, and a one-phase exponential association model was used with the following parameters: These results together with previously published data 2332 suggest that in vivo both TDG and MBD4 play a role in the removal of deaminated 5hmC residues. We investigated whether 5hmU, 5caC and 5fC residues are also substrates for the previously characterized bacterial and human DNA glycosylases.
When using the mono-functional DNA glycosylases, the samples after incubation were subjected to hot alkaline treatment. In agreement with previous observations all three human DNA glycosylases: Next, we examined the repair of 5caC and 5fC residues by bacterial and human enzymes. Despite being used in molar excess, none of the others E. These results indicate that hTDG is a main human enzyme removing carboxylated and formylated cytosines and confirms previous findings by other laboratories 20 Enzymatic activity of various E.
A DNA glycosylase activities of the E. In order to get insight into the structural bases of substrate specificity and catalytic mechanism of human MBD4, we performed crystallographic studies of MBD4 cat complexed with its DNA substrates.
For this purpose, a catalytically inactive MBD4 cat mutant has been generated. We also determined an unliganded structure at higher resolution 1. The flipped-out abasic site, thymine and 5hmU from 5hmU1 structure are well defined in electron density map into the enzyme active site pocket defined by residues —, —, Leu, Gly and Tyr Supplementary Figure S5. The bases superimpose well and make the same protein interactions Figure 4 B.
Their O4 and O2 atoms interact with the main chain amino group of Val and the Tyr side chain, respectively. Both N3 and O2 interact with the side chain of Gln The 5-hydroxymethyl group of 5hmU does not make protein interaction.
The mismatched thymine, AP site and 5hmU bases in productive, non-productive binding and mobile state are coloured pink, green, slate, yellow and orange, respectively.
N- and C-termini are indicated. C Residues in grey involved in the interactions with the orphan guanine pink and atom colours are labelled and shown as sticks.
MBD4 cat binds DNA via three regions the loops — and — and the Gly-rich hairpin loop of HhH motif — and interacts mostly with the strand containing the substrate base. The main chain carbonyl groups of Arg and Leu pack against the opposite guanine and provide specific hydrogen bonds to its N1 and N2 atoms Figure 4 C. Notably, Arg seems to have a key role in locking the flipped-out base in a productive binding for catalysis. In both 5hmU2 5hmU is in a non-productive state and 5hmU3 5hmU is disordered structures, Arg interacts with the O6 atom of the unpaired G.
The 5hmU2 structure reveals a flipped-out 5hmU located at the entrance of the active site pocket in a position incompatible with the presence of the catalytic residue Asp This bound cytosine acts as an inhibitor by interacting with the same protein residues as those involved in the flipped-out base recognition.
A crystal symmetric terminal cytosine in cyan is held in the active site pocket. The bound cytosine acts as an inhibitor.
Lebanese University – Faculty of Science
While this article was submitted for publication, Manvilla et al. Despite lower resolution of the crystal structure 2.
Our work describes the first crystal structures of the catalytic domain of MBD4 in complex with mismatched bases located at the centre of a mer DNA duplex. The thymine and 5hmU mispaired with guanine is extruded from the DNA helix and located in the enzyme active site. The structures revealed that MBD4 specifically recognizes thymine and 5hmU opposite a guanine Figure 4. Interestingly, a group such as 5-hydroxymethyl on C5 would have no effect on MBD4 ligand binding as there is no interaction between it and the enzyme.
Importantly, it appeared unlikely for a cytosine and oxidized 5mC bases to be trapped in the active site pocket of MBD4 cat due to the unfavourable environment of the main chain amino group of Val which would create a repulsive force directly towards their NH 2 group.
Previous studies have also shown that 5caC and 5fC residues are substrates only for mammalian TDG proteins Repair of 5caC and 5fC residues in other than mammal organisms was unknown. In this study, for the first time, we demonstrated that E.
At present, biological role of Mug-catalysed removal of 5mC derivatives is not clear, since bacteria lack genome-wide methylation and TETs enzymes.
Here, we examined the substrate specificity of the full-length human MBD4 protein and MBD4 cat towards 5hmU and other oxidized derivatives of 5mC in order to further define the biological relevance of these DNA glycosylases. Nevertheless, our enzymolofie data provide evidence for the role of MBD4 as an efficient back-up enzyme which can specifically act bicohimie densely methylated Eznymologie regions of chromosomal DNA, where deamination of 5mC and 5hmC is expected to be more frequent.
Although TDG is endowed with wider substrate specificity when compared with MBD4, it lacks a MBD domain and tends to associate with transcriptionally structuraale euchromatin 38 and non-methylated CpG islands to protect them from aberrant DNA methylation 23 Finally, the current crystal structures especially the 5hmU3 structure can be used as a template to develop inhibitors of MBD4 cat in the context of the active DNA demethylation process in human cells.
If not, repaired 5hmU can lead to mutation, therefore human cells hold three DNA glycosylases to ensure efficient repair of this extremely mutagenic derivative of 5mC residue. Furthermore, Smug1 -knockout mice show no obvious cancer predisposition phenotype possibly implying no increase in spontaneous mutation rate These observations point to prevalent role of MBD4 in spontaneous mutation prevention in vivo.
Indeed, it was shown that TDG is associated with transcriptionally active euchromatin 38whereas MBD4 rather localize in heterochromatin regions which is in general heavily methylated 78. Funding for open enzymlogie charge: National Center for Biotechnology InformationU.
Journal List Nucleic Acids Res v.
(Biochimie structurale et métabolique – Bonamy) –
Published online Jul Author information Article notes Copyright and License information Disclaimer. Correspondence may also be addressed to Alexander A. Published by Oxford University Press.
This article has been cited by other articles in PMC. Abstract Active DNA demethylation in mammals occurs via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine 5hmC by the ten-eleven translocation family of proteins TETs.
Crystallization and structure determination of MBD4 cat Crystallization conditions are summarized in Table 1. Crystallographic data and refinement parameters. Open in a separate window.
Values for the highest resolution shell are in parentheses. Determination of the kinetic parameters of DNA glycosylase activities To measure the kinetic parameters of DNA glycosylases-catalysed excision of modified bases, reactions were performed under single turnover conditions.
Activity of bacterial and human DNA glycosylases on oligonucleotides containing oxidized and deaminated derivatives of 5mC We investigated whether 5hmU, 5caC and 5fC residues are also substrates for the previously characterized bacterial and human DNA glycosylases. Crystal structures of ligand-free and substrate-bound MBD4 cat In order to get insight into the structural bases of substrate specificity and catalytic mechanism of human MBD4, we performed crystallographic studies of MBD4 cat complexed with its DNA substrates.
Conflict of interest statement. Supplementary Material Supplementary Data: Click here to view. Pfeifer GP, Besaratinia A.
Mutational spectra of human cancer. The enigmatic thymine DNA glycosylase. The intricate structural chemistry of base excision repair machinery: Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.
Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4: Mbd4 inactivation increases Cright-arrowT transition mutations and promotes gastrointestinal tumor formation. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd Mismatch repair in methylated DNA.