Journal: Nature Communications

Article Title: An Aurora B-RPA signaling axis secures chromosome segregation fidelity

doi: 10.1038/s41467-023-38711-2

Figure Lengend Snippet: HDX changes between RPA and RPA S384D are shown in the ( a ) absence or ( b ) presence of ssDNA (ssDNA is depicted in black). Changes in deuterium incorporation are observed in almost all DNA binding and protein-interaction domains. Data are mapped onto the structure of human RPA which is built using the structures of the OB domains from crystal structures. The regions colored yellow correspond to peptides that were not identified in the MS analysis of either or both the wild type and mutant RPA samples. The flexible linkers were modeled using AlphaFold. The position of Ser-384 is denoted in green. Data are presented as ±SDM from three independent experiments. Bio-layer interferometry analysis of RPA or RPA S384D binding to DSS1 in the ( c ) absence or ( d ) presence of ssDNA. RPA S384D shows reduced binding to DSS1 in the absence of DNA. When ssDNA-bound, almost complete loss of DSS1 binding to RPA is observed.

Article Snippet: A codon-optimized open reading frame for Human DSS1 (DSS1) was synthesized (Genscript Inc.) with a SUMO protease cleavable N-terminal MVKIH-Strep-6x-HIS-SUMO tag.

Techniques: Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: An Aurora B-RPA signaling axis secures chromosome segregation fidelity

doi: 10.1038/s41467-023-38711-2

Figure Lengend Snippet: During mitosis, RPA bound to ssDNA intermediates or free in solution is phosphorylated by Aurora B kinase at Ser-384 (DBD-B) in the large RPA70 subunit. We propose that phosphorylation releases the protein-interaction domains (OB-F or PID 70N and wh or PID 32C ) and promotes the formation of higher-density RPA-bound ssDNA filaments. The protein-interaction domains can then promote binding to RPA-interacting proteins. The feedback mechanism that modulates Aurora B activity through RPA phosphorylation and the involvement of R loops also remains to be elucidated. Since the site of phosphorylation resides close to the DSS1 binding site, recruitment of DSS1-BRCA2 is inhibited leading to suppression of homologous recombination during mitosis. Deregulation of the Aurora B-RPA signaling circuit leads to errors in chromosome segregation fidelity.

Article Snippet: A codon-optimized open reading frame for Human DSS1 (DSS1) was synthesized (Genscript Inc.) with a SUMO protease cleavable N-terminal MVKIH-Strep-6x-HIS-SUMO tag.

Techniques: Phospho-proteomics, Binding Assay, Activity Assay, Homologous Recombination

Journal: Protein expression and purification

Article Title: Rapid and Efficient Purification of Drosophila Homeodomain Transcription Factors for Biophysical Characterization

doi: 10.1016/j.pep.2019.02.001

Figure Lengend Snippet: Vector map (A) and sequence map (B) of HisSUMO-HD constructs. Each of the HDs for FTZ, ABD-A, ABD-B, ANTP, and UBX were cloned into a T7-inducible HisSUMO vector between KpnI and XbaI restriction sites. (C) Sequence alignment of overexpressed D. melanogaster HDs. The 107 member homeodomain family was submitted to ClustalW [48] using default values and analysed using Jalview [49]. Only the five HDs studied in this paper are shown. Highly conserved residues within similar physicochemical properties in all 107 HDs analysed are shown (red – positive, blue – negative, yellow – polar, green – hydrophobic). Absolutely conserved residues and residues with greater than 70% similarity are labelled with a star or cross, respectively.

Article Snippet: The HD open reading frames of FTZ (Uniprot: {"type":"entrez-protein","attrs":{"text":"P02835","term_id":"25453437","term_text":"P02835"}} P02835 , residues 254–313) and ANTP ( {"type":"entrez-protein","attrs":{"text":"P02833","term_id":"123317","term_text":"P02833"}} P02833 , residues 297–356) were Escherichia coli ( E. coli ) codon-optimized and synthesized by Synbio Technologies and cloned into a modified T7-inducible HisSUMO vector in between compatible Kpn I and Xba I restriction sites ( ).

Techniques: Plasmid Preparation, Sequencing, Construct, Clone Assay

Journal: Protein expression and purification

Article Title: Rapid and Efficient Purification of Drosophila Homeodomain Transcription Factors for Biophysical Characterization

doi: 10.1016/j.pep.2019.02.001

Figure Lengend Snippet: (A) Purification gel of ANTPHD showing samples taken at each step of the purification process (as described in the Materials & Methods). (B) Gel of purified D. melanogaster HDs for FTZ, ABD-A, ABD-B, ANTP, and UBX. Each lane was loaded with 5 ug of purified HD protein. Spectra Multicolor Broad Range Protein Ladder (Thermo-Fisher) was used in both gels to monitor migration and determine molecular weights of the HisSUMO-HD, HisSUMO tag, and isolated HD proteins. Gels were stained with Coomassie Brilliant Blue G-250 and visualized using an iBright FL1000 gel imager.

Article Snippet: The HD open reading frames of FTZ (Uniprot: {"type":"entrez-protein","attrs":{"text":"P02835","term_id":"25453437","term_text":"P02835"}} P02835 , residues 254–313) and ANTP ( {"type":"entrez-protein","attrs":{"text":"P02833","term_id":"123317","term_text":"P02833"}} P02833 , residues 297–356) were Escherichia coli ( E. coli ) codon-optimized and synthesized by Synbio Technologies and cloned into a modified T7-inducible HisSUMO vector in between compatible Kpn I and Xba I restriction sites ( ).

Techniques: Purification, Migration, Isolation, Staining

Journal: Protein expression and purification

Article Title: Rapid and Efficient Purification of Drosophila Homeodomain Transcription Factors for Biophysical Characterization

doi: 10.1016/j.pep.2019.02.001

Figure Lengend Snippet: EMSA of isolated FTZ, ANTP, and ABD- A HDs binding to the consensus sequence 5’-GCGTTTAATTAGCCC-3’ (A-C) and the null sequence 5’-GCGCGCCGGCGGCCC-3’ (D-F).

Article Snippet: The HD open reading frames of FTZ (Uniprot: {"type":"entrez-protein","attrs":{"text":"P02835","term_id":"25453437","term_text":"P02835"}} P02835 , residues 254–313) and ANTP ( {"type":"entrez-protein","attrs":{"text":"P02833","term_id":"123317","term_text":"P02833"}} P02833 , residues 297–356) were Escherichia coli ( E. coli ) codon-optimized and synthesized by Synbio Technologies and cloned into a modified T7-inducible HisSUMO vector in between compatible Kpn I and Xba I restriction sites ( ).

Techniques: Isolation, Binding Assay, Sequencing