Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities

Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5′ half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein–DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions

The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA

BACKGROUND: MBD5 and MBD6 are two uncharacterized mammalian proteins that contain a putative Methyl-Binding Domain (MBD). In the proteins MBD1, MBD2, MBD4, and MeCP2, this domain allows the specific recognition of DNA containing methylated cytosine; as a consequence, the proteins serve as interpreters of DNA methylation, an essential epigenetic mark. It is unknown whether MBD5 or MBD6 also bind methylated DNA; this question has interest for basic research, but also practical consequences for human health, as MBD5 deletions are the likely cause of certain cases of mental retardation. PRINCIPAL FINDINGS: Here we report the first functional characterization of MBD5 and MBD6. We have observed that the proteins colocalize with heterochromatin in cultured cells, and that this localization requires the integrity of their MBD. However, heterochromatic localization is maintained in cells with severely decreased levels of DNA methylation. In vitro, neither MBD5 nor MBD6 binds any of the methylated sequences DNA that were tested. CONCLUSIONS: Our data suggest that MBD5 and MBD6 are unlikely to be methyl-binding proteins, yet they may contribute to the formation or function of heterochromatin. One isoform of MBD5 is highly expressed in oocytes, which suggests a possible role in epigenetic reprogramming after fertilization

Le double jeu de l’épigénétique

Le mot « épigénétique » désigne les phénomènes transmis héréditairement, et qui modulent l’activité du génome sans changer sa séquence. Nous abordons le lien entre épigénétique et cancer par ces questions : quels sont les processus épigénétiques et comment sont-ils altérés dans les cancers ? Comment l’épigénétique se combinet- elle à la génétique pour entraîner un processus cancéreux ? Quelles sont les conséquences pour le diagnostic et la thérapie ? Quels liens entre épigénétique et environnement ? Nous arriverons à la conclusion que les changements épigénétiques sont à la fois cibles et acteurs dans le processus tumora

MBD4 cooperates with DNMT1 to mediate methyl-DNA repression and protects mammalian cells from oxidative stress

International audienc

Identification of an Alu-mediated 5.7-kb deletion of the LU gene in a pregnant Moroccan woman with anti-Lu3.

International audienc

MBD5 and MBD6 can colocalize with methylated regions in mouse nuclei.

<p>The indicated proteins were transfected into mouse 3T3 cells. The distribution of the various proteins in fixed cells was recorded, and representative images are provided. When different types of localization occurred, their approximate proportion is indicated. A-MBD5 isoform 1 always colocalizes with the chromocenters, DAPI-dense regions that harbor hypermethylated heterochromatin; mutating either MBD or PWWP domain changes this distribution to a diffuse nuclear pattern. Isoform 2 is also incapable of chromocenter localization. B- MBD6 colocalizes with chromocenters in 25% of the observed cells. Upon deletion of the MBD, the pattern becomes diffuse in all cells.</p

qRT-PCR primers used in this study.

<p>qRT-PCR primers used in this study.</p

MBD5 and MBD6 are differentially expressed in mouse tissues.

<p>A-MBD5 Isoform 1 is expressed at highest levels in the brain, whereas isoform 2 is most expressed in oocytes. B- MBD6 is expressed at highest levels in the testis. Expression levels were measured by quantitative RT-PCR, with normalization to the RPS16 gene.</p

MBD5 and MBD6 can be recruited to chromocenters in <i>Dnmt1</i>−/− cells.

<p>The various GFP fusions were transfected into mouse fibroblasts of the indicated genotype. A -ZBTB4, MBD5, and MBD6, are recruited to the chromocenters of <i>Dnmt1</i>+/+ cells. B- MBD5 and MBD6 can be recruited to the demethylated chromocenters of <i>Dnmt1</i>−/− cells; ZBTB4 does not overlap with the chromocenters in the same cells.</p