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In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

RecA protein is essential to general genetic recombination in Escherichia coli. In the presence of ATP, a stoichiometric amount of recA protein forms D-loops from superhelical closed-circular DNA (form I DNA) and homologous single-stranded fragments, and subsequently dissociates the D-loops. Under appropriate conditions, the hydrolysis of ATP by recA protein depends on the presence of both double-stranded DNA and homologous single-stranded fragments (homology-dependent hydrolysis). In the presence of form I DNA, most of the homology-dependent hydrolysis of ATP by recA protein is related to the dissociation of D-loops rather than the formation of D-loops. RecA protein also promoted the homology-dependent hydrolysis of ATP in the presence of nicked-circular DNA (form II DNA), but unlike the case of form I DNA, this hydrolysis was associated with an increase in the amount of mature D-loops that were detected by the D-loop assay. When double-stranded DNA was superhelical, the homology-dependent hydrolysis of ATP continued at the same rate even after all the D-loops were dissociated. This correlates with our earlier observation that in the process of formation and dissociation of D-loops, form I DNA was converted to an inactive substrate without any apparent damage to the DNA, probably by the formation of a complex with recA protein. All of the observations described above can be explained by a model in which a common mechanism causes dissociation of D-loops from form I DNA, inactivation of form I DNA, and growth of D-loops in form II DNA. The mechanism might involve cooperative binding of recA protein to the duplex DNA from the site of the nascent D-loop, resulting in unidirectional unwinding of the duplex DNA.

Truncated templates of a cloned actin gene (actin 5) of Dictyostelium discoideum were transcribed in a HeLa cell extract from a position indistinguishable from the in vivo initiation site. The efficiency of this accurate transcription, however, was very low compared with that of the adenovirus 2 major late gene in the same system. Transcription of the actin 6 and 8 genes, of which the 5'-flanking regions differ in their base sequences from the corresponding region of the actin 5 gene, was not good in either fidelity or efficiency. When the partially purified RNA polymerase II of D. discoideum was used instead of the HeLa cell extract, no accurate transcription of the actin 5 gene occurred. However, simultaneous addition of RNA polymerase II and HeLa cell extract into the cell-free system caused a shift of the in vitro initiation point of transcription of the actin 5 gene to a position about 20 nucleotides downstream from the presumed in vivo initiation site. The reason for the shift remains unknown, but it is supposed that some factor(s) other than RNA polymerase II are involved in the accurate initiation of transcription and the maintenance of transcription efficiency of the actin genes.

The template specificity of DNA-dependent RNA polymerases I and II (ribonucleoside 5'-triphosphate : RNA nucleotidyltransferase [EC 2.7.7.6]) of Dictyostelium discoideum was investigated with several synthetic polynucleotides at three different stages of development. Both the enzymes exhibited several common characteristics for some templates, and distinctly different properties for other ones. Of single-stranded homopolymers, the strands of pyrimidine nucleotides were much transcribed in the order of poly(dC) greater than poly(dT). The double-stranded homopolymers, poly(dA). poly(dT)) and poly(dG).poly(dC) were transcribed asymmetrically, the pyrimidine-containing strand being preferentially read. Transcription of double-stranded alternating copolymers, poly([d(A-T)].poly[d(A-T)] and poly[d(G-C)].poly[d(g-C)] occurred to some extent. Except for poly(rC), all of the single-stranded ribonucleotide homopolymers were extremely poor as templates. The polynucleotides containing thymidine were more efficient templates for polymerase I than polymerase II. The enzyme activities of the two polymerases were more or less variable with some polynucleotides among three stages of development, suggesting the possibility that D. discoideum RNA polymerases tend to change their template specificity during development.