Additional file 1 of Archaeal Orc1 protein interacts with T-rich single-stranded DNA
Creators: name: Wegrzyn, Katarzyna
name: Konieczny, Igor
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: 29999 Physical Sciences not elsewhere classified
subject: FOS: Physical sciences
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Biotechnology
subject: Cancer
subject: 60506 Virology

description: Additional file 1: Figure S1. Sequence alignment of archaeal origin regions. The sequences of origin regions of A. pernix (NC_000854), Pyrococcus abyssi (NC_000868), Pyrococcus furiosus (NC_003413) and Sulfolobus solfataricus (NC_003106) were chosen based on the work of Robinson and co-authors [26]. The sequence alignment was done with the use of Clustal Omega tool [27]. The sequences of the Orc proteins’ binding sites (ORB sites) are bolded. Figure S2. Purification of ApOrc1 protein. The ApOrc1 protein was purified as a His6-tagged variant from the E. coli Rosetta LysS strain. After overproduction of ApOrc1 protein and cells lysis, a fraction of soluble proteins was applied onto a Ni–NTA resin. After the washing step, the protein bound to the resin was eluted with buffer containing 250 mM EDTA (see Materials and Methods). 5 µl of collected fractions were mixed with Laemmli buffer and separated in 12.5% polyacrylamide gel stained with Coomassie Blue dye. M—PageRuler™ Prestained Protein Ladder (Thermo Scientific) (lane 1); S—fraction of soluble proteins (lane 2); FT- fraction of proteins that did not bind to the resin (line 3); E- fractions collected during elution of proteins from the resin (lanes 4–8). Figure S3. Binding of Ori1 region by ApOrc1 protein. Analysis of ApOrc1 protein binding to dsDNA with EMSA technique. Binding to dsDNA fragments containing sequence of Ori1 region (A) or non-specific sequence (fragment of pUC18 plasmid) (B) was tested. (A, B) Fluorescently labeled dsDNA fragment (1 pmol) was incubated with increasing concentration of ApOrc1 protein (100, 200, 300 nM) and then separated in 5% PAGE in TBE buffer. Black arrows indicate the nucleoprotein complexes (A). Black asterisk indicates fuzzy band of unspecific nucleoprotein complex (B). Figure S4. Binding of E. coli SSB protein to ssDNA fragments containing sequence of top and bottom strand of the right part of the AT-rich region of Ori1. Binding was analyzed with SPR technique. Biotinylated ssDNA fragments (Additional file 1: Table S1) were immobilized on a surface of sensor chip SA. Injections containing the indicated concentrations of SSB protein in HBS-EP buffer were performed. HBS-EP was also used as a running buffer. Figure S5. Binding of ApOrc1 protein to ssDNA fragments containing sequence of top and bottom strand of right part of the AT-rich region of Ori1. Binding of the ApOrc1 protein to indicated ssDNA fragments containing sequence of right part of the AT-rich region of Ori1 was analyzed with SPR technique. Biotinylated ssdna fragments (Additional file 1: Table S1) were immobilized on a surface of sensor chip SA. Injections containing the indicated concentrations of protein in HBS-EP buffer were performed. HBS-EP was also used as a running buffer. Figure S6. Influence of nucleotides on ApOrc1 protein binding to ssDNA. Binding of the ApOrc1 protein to ssDNA fragments (Additional file 1: Table S1) containing right bottom strands’ sequence of the Ori1 AT-rich region was analyzed with SPR technique. Biotinylated ssDNA fragments were immobilized on a surface of SA sensor chip. Injections containing the indicated concentrations of protein in HBS-EP buffer with (A) or without (B) ATP, with AMP-PMP (non-hydrolyzable analogue of ATP) (C) and ADP (D) were performed. HBS-EP was used as a running buffer. Figure S7. Binding of ApOrc1 protein to non-specific ssDNA fragment. Binding of ApOrc1 protein to ssDNA fragments (Additional file 1: Table S1) containing sequence of AT-rich region of plasmid RK2 was analyzed with SPR technique. Biotinylated ssDNA fragments were immobilized on a surface of sensor chip SA. Injections containing the indicated concentrations of protein in HBS-EP buffer were performed. HBS-EP was also used as a running buffer. Figure S8. MST analysis of ApOrc1 binding to ssDNA fragments. Binding was analyzed with MST technique. Fluorescently labeled ssDNA fragments containing sequence of polyT(70) (A, blue) and poly A(70) (A, red) or sequence of R bottom (B, blue) and R top strands (B, red) of AT-rich region of Ori1 (Table S1) were mixed with increasing concentration of ApOrc1 protein in EDBS buffer. Mixtures were transferred to Standard Monolith NT™ Capillaries and fluorescence was measured with Monolith NT.115. For each ssDNA fragment, at least three independent experiments were performed. The obtained data were analyzed with MO. Affinity Analysis software and presented as dependence of change in the fluorescence from protein concentration. Figure S9. Binding of polyT ssDNA by ApOrc1 protein. Binding of ApOrc1 protein to homopolymer of thymidine (polyT(70)) was analyzed with EMSA technique. Fluorescently labeled ssDNA fragment (1 pmol) was incubated with increasing concentration of ApOrc1 protein (100, 200, 300 nM) and then separated in 5% PAGE in TBE buffer. Black arrow indicates the nucleoprotein complex. Black asterisks indicates position of dyes from DNA loading buffer: xylene cyanol (*), bromophenol blue (**). Figure S10. Dependence of ApOrc1 binding to R bottom ssDNA fragments from ATP concentration. Binding was analyzed with SPR technique. Biotinylated ssDNA fragments containing sequence of R bottom strand of AT-rich region of Ori1 (Additional file 1: Table S1) were immobilized on a surface of sensor chip SA. Injections containing the constant concentrations of ApOrc1 (125 nM) protein in HBS-EP buffer supplemented with increasing concentration of ATP were performed. HBS-EP was also used as a running buffer. Table S1. Oligonucleotides used in this study.