Single-Molecule Analysis of Replication Protein A–DNA Interactions

DNA-binding mechanism and evolution of Replication Protein A

“…In all forms of life, coating and protecting exposed ssDNA from nucleases is essential (Oliveira, 2021). In Eukarya, RPA has been shown to be an abundant multi-domain heterotrimeric protein complex, which is essential to all DNA processing events in DNA replication, recombination and repair (Bain et al, 2018; Brosey et al, 2013; Dueva and Iliakis, 2020). Here, we explored the quaternary structure of PabRPA and the conformational changes that occur upon DNA-binding.…”

DNA-binding mechanism and evolution of Replication Protein A

“…In all forms of life, coating and protecting exposed ssDNA from nucleases is essential (Oliveira, 2021). In Eukarya, RPA has been shown to be an abundant multi-domain heterotrimeric protein complex, which is essential to all DNA processing events in DNA replication, recombination and repair (Bain et al, 2018; Brosey et al, 2013; Dueva and Iliakis, 2020). Here, we explored the quaternary structure of PabRPA and the conformational changes that occur upon DNA-binding.…”

G-quadruplex–stalled eukaryotic replisome structure reveals helical inchworm DNA translocation

“…The RPA expression plasmid, p11d-tRPA, was a gift from Marc Wold (University of Iowa). RPA was purified as reported previously ( 69 ). Eight liters of BL21-Codonplus DE3 (RIL) transformed with RPA expression plasmid were grown at 37°C to OD 600 = 0.8.…”

Single-molecule analysis of PARP1-G-quadruplex interaction

“…The method for conducting single-molecule TIRFM PARP1-DNA binding experiments is as follows and includes the DNA substrates listed ( Supplementary Table 1 ). Prior to the experiment, quartz slides (25 mm × 75 mm × 1 mm #1x3x1MM, G. Frinkenbeiner, Inc., Waltham, MA, USA) and cover glass (24 mm × 60 mm-1.5, Fisherbrand, Fisher Scientific, Hampton, NH, USA) are treated with passivation and PEGylation (36). The flow cell is treated with 0.2 mg/mL NeutrAvidin (#3100, Thermo Fisher Scientific, Waltham, MA, USA) to tether biotinylated molecules to the flow cell surface.…”

DNA-binding mechanism and evolution of Replication Protein A

“…In all forms of life, coating and protecting exposed ssDNA from nucleases is essential (Oliveira, 2021). In Eukarya, RPA has been shown to be an abundant multi-domain heterotrimeric protein complex, which is essential to all DNA processing events in DNA replication, recombination and repair (Bain et al, 2018; Brosey et al, 2013; Dueva and Iliakis, 2020). Here, we explored the quaternary structure of PabRPA and the conformational changes that occur upon DNA-binding.…”

G-quadruplex–stalled eukaryotic replisome structure reveals helical inchworm DNA translocation

“…The RPA expression plasmid, p11d-tRPA, was a gift from Marc Wold (University of Iowa). RPA was purified as reported previously ( 69 ). Eight liters of BL21-Codonplus DE3 (RIL) transformed with RPA expression plasmid were grown at 37°C to OD 600 = 0.8.…”

Single-molecule analysis of PARP1-G-quadruplex interaction

“…The method for conducting single-molecule TIRFM PARP1-DNA binding experiments is as follows and includes the DNA substrates listed ( Supplementary Table 1 ). Prior to the experiment, quartz slides (25 mm × 75 mm × 1 mm #1x3x1MM, G. Frinkenbeiner, Inc., Waltham, MA, USA) and cover glass (24 mm × 60 mm-1.5, Fisherbrand, Fisher Scientific, Hampton, NH, USA) are treated with passivation and PEGylation (36). The flow cell is treated with 0.2 mg/mL NeutrAvidin (#3100, Thermo Fisher Scientific, Waltham, MA, USA) to tether biotinylated molecules to the flow cell surface.…”

DNA-binding mechanism and evolution of Replication Protein A

“…In all forms of life, coating and protecting exposed ssDNA from nucleases is essential (Oliveira, 2021). In Eukarya, RPA has been shown to be an abundant multi-domain heterotrimeric protein complex, which is essential to all DNA processing events in DNA replication, recombination and repair (Bain et al, 2018; Brosey et al, 2013; Dueva and Iliakis, 2020). Here, we explored the quaternary structure of PabRPA and the conformational changes that occur upon DNA-binding.…”

G-quadruplex–stalled eukaryotic replisome structure reveals helical inchworm DNA translocation

“…The RPA expression plasmid, p11d-tRPA, was a gift from Marc Wold (University of Iowa). RPA was purified as reported previously ( 69 ). Eight liters of BL21-Codonplus DE3 (RIL) transformed with RPA expression plasmid were grown at 37°C to OD 600 = 0.8.…”

Single-molecule analysis of PARP1-G-quadruplex interaction

“…The method for conducting single-molecule TIRFM PARP1-DNA binding experiments is as follows and includes the DNA substrates listed ( Supplementary Table 1 ). Prior to the experiment, quartz slides (25 mm × 75 mm × 1 mm #1x3x1MM, G. Frinkenbeiner, Inc., Waltham, MA, USA) and cover glass (24 mm × 60 mm-1.5, Fisherbrand, Fisher Scientific, Hampton, NH, USA) are treated with passivation and PEGylation (36). The flow cell is treated with 0.2 mg/mL NeutrAvidin (#3100, Thermo Fisher Scientific, Waltham, MA, USA) to tether biotinylated molecules to the flow cell surface.…”