Background: Although highly homologous to MBD2, the functional role of MBD3 remains in question. Results: MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated CpG dinucleotides. Conclusion: Dynamic distribution between methylated and unmethylated sites modifies the genomic localization of MBD3. Significance: Changes in the dynamic distribution on DNA dictate functional differences between MBD proteins.
Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference formCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to mCpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain.
DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution.
In contrast to the narrowing of options in academic careers, the bioscience industry offers robust employment opportunities for STEM-trained workers, especially those who display both scientific and business talent. Unfortunately, traditional science programs typically lack curricular features that develop this type of worker. The North Carolina State University Master of Microbial Biotechnology (MMB) program facilitates industry-specific experiential learning to fill this training gap. Similar programs often rely on a single industry internship to provide students relevant work experience, but completion of one internship might not suffice to position students for employment in a highly competitive job market. The MMB program requires students to complete an internship and three practicum projects in an industry setting, to promote development of key skills in a variety of areas, to build confidence in the ability to perform initial job duties, and to establish a more extensive work history in industry. In this Perspective we discuss an unmet need in undergraduate and graduate STEM education that can be filled by incorporating a similar set of industry-specific work experiences for students who desire to transition from academe into the life science industry.
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