Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Replication Protein A (RPA) Regulates Its DNA Binding Activity through Redox

  • Published : 20001200
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Abstract

Keywords

References

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  9. Yeast wild-type RPA and human mutant RPA were puri-fied according to the Reference 2 and 8, respectively.
  10. RPA-ssDNA binding assay: Oligo $(dT)_{50}$was 5'-end label-ed with $[γ^32P]$ATP (Du Pont) and T4 polynucleotide kin-ase (USB) based on the manufacturer’s instructions. The indicated amount of wild-type or mutant RPA was incu-bated with 100 fmol of $5'-^32$P-labeled Oligo $(dT)_{50}$ at room temperature for 15 min in the reaction mixtures (30 mL) containing 50 mM Hepes-KOH (pH 7.8), 10 mM $MgCl_2$ polydI : dC (0.2 mg), BSA (0.2 μg/μL), and indicated amounts of DTT or NaDI. Protein-DNA complexes were analyzed using 5% polyacrylamide gels in 1x TBE buffer (acrylamide: bisacrylamide = 79 : 1), The gels were dried and exposed to x-ray films (Kodak), The bands of interest were excided from the gels and measured for radioactivity using a Beckman Scintillation Counter LS 6500
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  16. Plasmid constructs and site-directed mutagenesis: cDNA encoding the p70 subunit ofhuman RPA was cloned into baculovirus transfer vector pVL941-SW. For the change Cys-289’ Ala, the plasmid pVL941SW-70 (carring wild-type p70 gene) and the fellowing primers were used in the PCR reaction, with the underlined nucleotides changes from the wild-type sequence: 5-TCC GTC ATG CCC GCC GAG GAC GAC CAT-3 and 5-ATG GTC GTC CTC GGC GGG CAT GAC GGA-3. The mutation procedure was performed according to the Stratagene protocol.
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Cited by

  1. Identification of the Structural Change of Human Replication Protein A (hRPA) in the ssDNA Binding and Redox Potential vol.26, pp.8, 2000, https://doi.org/10.5012/bkcs.2005.26.8.1168