Cloning of the large subunit of replication protein A (RPA) from yeast Saccharomyces cerevisiae and its DNA binding activity through redox potential.
Eukaryotic replication protein A (RPA) is a single-stranded(ss) DNA binding protein with multiple functions in DNA replication, repair, and genetic recombination. The 70-kDa subunit of eukaryotic RPA contains a conserved four cysteine-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Recently, we described a novel function for the zinc-finger motif in the regulation of human RPA's ssDNA binding activity through reduction-oxidation (redox). Here, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its RPA32 and/or RPA14 subunits. Yeast RPA requires a reducing agent, such as dithiothreitol (DTT), for its ssDNA binding activity. Also, under non-reducing conditions, its DNA binding activity decreases 20 fold. In contrast, the RPA70 subunit does not require DTT for its DNA binding activity and is not affected by the redox condition. These results suggest that all three subunits are required for the regulation of RPA's DNA binding activity through redox potential.
Jeong
Haeng-Soon
HS
Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Pusan 609-735, Korea.
Jeong
In-Chel
IC
Kim
Andre
A
Kang
Shin-Won
SW
Kang
Ho Sung
HS
Kim
Yung-Jin
YJ
Lee
Suk-Hee
SH
Park
Jang-Su
JS
D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004229:Dithiothreitol; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010084:Oxidation-Reduction; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016335:Zinc Fingers
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D021122:Protein Subunits; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine; D004229:Dithiothreitol