Xu, S. y. | Search bioRxiv

bioRxiv2024

Distinct structural and functional heterochromatin partitioning of lamin B1 and B2 revealed using genome-wide Nicking Enzyme Epitope targeted DNA sequencing.

A genome-wide chromatin profiling technology, named as Nicking Enzyme Epitope targeted DNA sequencing (NEED-seq) in which antibody-targeted controlled nicking by Nt.CviPII-pGL is used to study specific protein-DNA complexes. NEED-seq is performed in situ in formaldehyde fixed cells, allowing for both visual and genomic resolution of epitope bound chromatin. When applied to nuclei, NEED-seq yielded genome-wide chromatin associated proteins and histone post-translational modifications (PTMs). NEED-seq of lamin B1/B2 demonstrated their association with heterochromatin. Lamin B1 and B2 associated domains (LAD) segregated to three different states, and states with stronger LAD correlated with heterochromatic marks. Hi-C analysis displayed A and B compartment with equal lamin B1/B2 distribution, although methylated DNA remained high in B compartment. LAD clustering with Hi-C resulted in subcompartments, with lamin B1-B2 partitioning to facultative and constitutive heterochromatin respectively and were associated with neuronal development. Thus, lamin B1 and B2 have structural and functional partitioning in mammalian nucleus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/603107v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@44c36forg.highwire.dtl.DTLVardef@7ab1adorg.highwire.dtl.DTLVardef@126e99eorg.highwire.dtl.DTLVardef@1b38bd7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Graphical abstract: Model depicting association of lamin B1 and B2 in A (facultative heterochromatin) and B (constitutive heterochromatin) compartmentalization. C_FIG

genomics↗

bioRxiv2023

Type II restriction of 2-aminoadenine (dZ) modified DNA and production of dZ-modified plasmid in E. coli

The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Herein we report the results of 147 REases activity on dZ-modified PCR DNA. Among the enzymes tested, 53.1% are resistant or partially resistant, and 46.9% are sensitive when the restriction sites contain 1 to 6 modified bases. Sites with 4-6 dZ substitutions are most likely resistant to Type II restriction. Our results support the notion that dZ-modified phage genomes are evolved to combat host- encoded restriction systems. dZ-modified DNA can also "slow down" phage T5 exonuclease degradation, but it has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into E. coli plasmid, dZ incorporation level reached 19-20% dZ/(dZ+dA). dZ level can be further increased to 28.9-44.3% with co-expression of a DNA polymerase gene from the same phage. High level of dZ incorporation in recombinant plasmid is possible by co-expression of purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (single-stranded DNA binding protein SSB). This work has a general interest for molecular biologists working on dZ DNA modification and restriction systems. It provides a foundation for future research on screening dZ-dependent Type IV restriction systems. The results presented herein may have implication in gene therapy utilizing dZ-modified DNA, provided that human RNA polymerase variants can efficiently perform transcription from a dZ-modified template.

microbiology↗