Journal: Chemical research in toxicology

Article Title: Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.

doi: 10.1021/acs.chemrestox.2c00172

Figure Lengend Snippet: Figure 7. 3′-terminus produced by hyTDG-lyase cannot be extended by DNA Pol β but is resolved by APE1. In lane 1, A 5′-FAM-labeled 79 base oligonucleotide containing a U in a U:G mispair (2.5 pmol, 0.2 μM). In lane 2, the oligonucleotide was incubated with UDG (2.5 U, 0.84 pmol, 0.07 μM) for 1 h at 37 °C in CutSmart buffer, and subsequently, the abasic site was cleaved by incubating with APE1 (5 U, 0.18 pmol, 0.01 μM) for an additional 30 min. In lane 3, Pol β (6.2 pmol, 0.5 μM) and E. coli ligase (5 U, 4 pmol, 0.32 μM), dCTP (250 pmol, 20 μM), and NAD+ (325 pmol, 26 μM) were added for an additional 1 h to simulate short-patch BER. Lane 4 was otherwise identical to lane 2, but hyTDG-lyase was used instead of APE1 at 37 °C for 30 min. Lane 5 was otherwise identical to lane 3, except hyTDG-lyase (26.9 pmol, 2.15 μM) was used instead of APE1 at 37 °C. In lane 6, we similarly generated an abasic site that was then cleaved by hyTDG-lyase for 30 min at 37 °C. Then APE1, Pol β, dCTP, and E. coli ligase were added and incubated for an additional 1 h. Lane 6 demonstrates that the AP endonuclease activity of APE1 can clean up the PUA-βME 3′-end produced by the hyTDG-lyase. The complementary strand is labeled with a 5′-Cy5 fluorophore (red). The overlap between the full-length 5′-FAM-labeled oligo and its 5′-Cy5 complement is depicted as yellow.

Article Snippet: Human 8-oxoguanine DNA glycosylase (hOGG1, #NBP1-453180.1mg) and DNA polymerase β (Pol β, #NBP1-72434-0.5mg) were purchased from Novus Biologicals.

Techniques: Produced, Labeling, Incubation, Generated, Activity Assay

Journal: Cancers

Article Title: An Inducible BRCA1 Expression System with In Vivo Applicability Uncovers Activity of the Combination of ATR and PARP Inhibitors to Overcome Therapy Resistance

doi: 10.3390/cancers18020309

Figure Lengend Snippet: ( A ) ( Left panel ): dose–response curves of olaparib in colony formation assays in tetracycline-repressor (TR) expressing MDAMB436 cells with or without RAD51B complementation (MDAMB436-B-TR and MDAMB436-TR, respectively) and with (+BRCA1) or without (+LacZ) BRCA1 complementation. ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib for each cell line. ( B ) ( Left panel ) : dose–response curves of olaparib in colony formation assays in MDAMB436-B-TR cells expressing full-length BRCA1 (+FL), no BRCA1 (+LacZ), or different BRCA1 hypomorphs (+RING-less, +∆exon11, +BRCT-less). ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib for each cell line. ( C ) ( Left panel ): dose–response curves of olaparib in survival assays in MDAMB436-B-TR cells expressing full-length BRCA1 (+FL), no BRCA1 (+LacZ), or different BRCA1 hypomorphs (+C64R, +L1407P, +R1699Q). ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib for each cell line. Data for BRCA1 hypomorphs with deletions in the RING (RING-less, M48 START) or BRCT (BRCT-less) domains are included for comparative purposes. ( D ) ( Left panel ): dose–response curves of carboplatin in survival assays in MDAMB436-B-TR cells expressing full-length BRCA1 (+FL), no BRCA1 (+LacZ) or different BRCA1 hypomorphs (+RING-less, +∆exon11, +BRCT-less, +CC-mutant). ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of carboplatin for each cell line. ( E ) Western blot of immunoprecipitation experiments in MDAMB436-B-TR cells expressing full-length (+FL) or ∆exon11 BRCA1 and exposed to different doxycycline doses. GAPDH was used as a loading control. I = input; F = FLAG immunoprecipitation. ( F ) Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib at the different doses of doxycycline used. All data are from at least 3 biological replicates. Statistical analysis performed using One-Way ANOVA with Holm–Sidak multiple comparisons, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CFU = colony-forming units. Dotted lines in the olaparib IC50 graphs represent the minimal free concentration of olaparib (approximately 300 nM) in the plasma of patients on the established monotherapy dose of 300 mg twice daily. The original western blot figures can be found in .

Article Snippet: Plasmids containing the human BRCA1 (NM_007294) and RAD51B (NM_133509) open reading frames (ORFs) were obtained from Genscript (Ohu18572D) and OriGene (RC206457L3), respectively.

Techniques: Expressing, Concentration Assay, Mutagenesis, Western Blot, Immunoprecipitation, Control, Clinical Proteomics