Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA

ABSTRACT IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

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Nucleocytoplasmic Shuttling of Endocytic Proteins

The Journal of Cell Biology ◽

10.1083/jcb.153.7.1511 ◽

2001 ◽

Vol 153 (7) ◽

pp. 1511-1518 ◽

Cited By ~ 77

Author(s):

Manuela Vecchi ◽

Simona Polo ◽

Viviane Poupon ◽

Jan-Willem van de Loo ◽

Alexandre Benmerah ◽

...

Keyword(s):

Transcriptional Regulation ◽

Nuclear Export ◽

Myeloid Leukemia ◽

Nuclear Translocation ◽

Initial Rate ◽

Nucleocytoplasmic Shuttling ◽

Cellular Processes ◽

Transactivation Assay ◽

Ordered Assembly ◽

Endocytic Proteins

Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and α-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.

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Template‐Directed Copying of RNA by Non‐enzymatic Ligation

Angewandte Chemie ◽

10.1002/ange.202004934 ◽

2020 ◽

Vol 132 (36) ◽

pp. 15812-15817 ◽

Cited By ~ 2

Author(s):

Lijun Zhou ◽

Derek K. O'Flaherty ◽

Jack W. Szostak

Keyword(s):

Enzymatic Ligation

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Deciphering the rules governing assembly order of mammalian septin complexes

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0253 ◽

2011 ◽

Vol 22 (17) ◽

pp. 3152-3164 ◽

Cited By ~ 114

Author(s):

Mikael E. Sellin ◽

Linda Sandblad ◽

Sonja Stenmark ◽

Martin Gullberg

Keyword(s):

Mammalian Cells ◽

Temporal Order ◽

Lateral Diffusion ◽

Diffusion Barriers ◽

Generic Model ◽

Molecular Scaffolds ◽

Domain Interactions ◽

Ordered Assembly ◽

A Minor ◽

Combined Data

Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9–17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals interdependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six- to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.

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Interactions among CotB, CotG, and CotH during Assembly of the Bacillus subtilis Spore Coat

Journal of Bacteriology ◽

10.1128/jb.186.4.1110-1119.2004 ◽

2004 ◽

Vol 186 (4) ◽

pp. 1110-1119 ◽

Cited By ~ 53

Author(s):

Rita Zilhão ◽

Mónica Serrano ◽

Rachele Isticato ◽

Ezio Ricca ◽

Charles P. Moran ◽

...

Keyword(s):

Bacillus Subtilis ◽

Reverse Genetics ◽

Spore Coat ◽

Yeast Two Hybrid ◽

Bacillus Subtilis Spore ◽

Yeast Two Hybrid System ◽

Ordered Assembly ◽

Heterologous Antigens ◽

Two Hybrid ◽

Good Agreement

ABSTRACT Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides. One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface. The cotB gene was initially identified by reverse genetics as encoding an abundant coat component. cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66). Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46). Expression of cotB in sporulating cells of B. subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66. These results suggest that soon after synthesis, CotB undergoes a posttranslational modification. Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci. We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants. Therefore, both cotH and cotG are required for the efficient conversion of CotB-46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat. We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG. Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG. We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact. We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB.

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