Stopped-flow kinetic analysis of replication protein A-binding DNA. Damage recognition and affinity for single-stranded DNA reveal differential contributions of kon and koff rate constants

Steve M. Patrick, John Turchi

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47 Citations (Scopus)

Abstract

Replication protein A (RPA) is a heterotrimeric protein required for many DNA metabolic functions, including replication, recombination, and nucleotide excision repair (NER). We report the pre-steady-state kinetic analysis of RPA-binding DNA substrates using a stopped-flow assay to elucidate the kinetics of DNA damage recognition. The bimolecular association rate, kon, for RPA binding to duplex DNA substrates is greatest for a 1,3d(GXG), intermediate for a 1,2d(GpG) cisplatin-DNA adduct, and least for an undamaged duplex DNA substrate. RPA displays a decreased kon and an increased koff for a single-stranded DNA substrate containing a single 1,2d(GpG) cisplatin-DNA adduct compared with an undamaged DNA substrate. The kon for RPA-binding single-stranded polypyrimidine sequences appears to be diffusion-limited. There is minimal difference in kon for varying length DNA substrates; therefore, the difference in equilibrium binding affinity is mainly attributed to the koff. The kon for a purine-rich 30-base DNA is reduced by a factor of 10 compared with a pyrimidine-rich DNA of identical length. These results provide insight into the mechanism of RPA-DNA binding and are consistent with RPA recognition of DNA-damage playing a critical role in NER.

Original languageEnglish (US)
Pages (from-to)22630-22637
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number25
DOIs
StatePublished - Jun 22 2001
Externally publishedYes

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Replication Protein A
Single-Stranded DNA
Protein Binding
Rate constants
Kinetics
DNA
Substrates
DNA Repair
DNA Damage
A-Form DNA
Repair
Nucleotides
Genetic Recombination
Proteins
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Stopped-flow kinetic analysis of replication protein A-binding DNA. Damage recognition and affinity for single-stranded DNA reveal differential contributions of kon and koff rate constants",
abstract = "Replication protein A (RPA) is a heterotrimeric protein required for many DNA metabolic functions, including replication, recombination, and nucleotide excision repair (NER). We report the pre-steady-state kinetic analysis of RPA-binding DNA substrates using a stopped-flow assay to elucidate the kinetics of DNA damage recognition. The bimolecular association rate, kon, for RPA binding to duplex DNA substrates is greatest for a 1,3d(GXG), intermediate for a 1,2d(GpG) cisplatin-DNA adduct, and least for an undamaged duplex DNA substrate. RPA displays a decreased kon and an increased koff for a single-stranded DNA substrate containing a single 1,2d(GpG) cisplatin-DNA adduct compared with an undamaged DNA substrate. The kon for RPA-binding single-stranded polypyrimidine sequences appears to be diffusion-limited. There is minimal difference in kon for varying length DNA substrates; therefore, the difference in equilibrium binding affinity is mainly attributed to the koff. The kon for a purine-rich 30-base DNA is reduced by a factor of 10 compared with a pyrimidine-rich DNA of identical length. These results provide insight into the mechanism of RPA-DNA binding and are consistent with RPA recognition of DNA-damage playing a critical role in NER.",
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AB - Replication protein A (RPA) is a heterotrimeric protein required for many DNA metabolic functions, including replication, recombination, and nucleotide excision repair (NER). We report the pre-steady-state kinetic analysis of RPA-binding DNA substrates using a stopped-flow assay to elucidate the kinetics of DNA damage recognition. The bimolecular association rate, kon, for RPA binding to duplex DNA substrates is greatest for a 1,3d(GXG), intermediate for a 1,2d(GpG) cisplatin-DNA adduct, and least for an undamaged duplex DNA substrate. RPA displays a decreased kon and an increased koff for a single-stranded DNA substrate containing a single 1,2d(GpG) cisplatin-DNA adduct compared with an undamaged DNA substrate. The kon for RPA-binding single-stranded polypyrimidine sequences appears to be diffusion-limited. There is minimal difference in kon for varying length DNA substrates; therefore, the difference in equilibrium binding affinity is mainly attributed to the koff. The kon for a purine-rich 30-base DNA is reduced by a factor of 10 compared with a pyrimidine-rich DNA of identical length. These results provide insight into the mechanism of RPA-DNA binding and are consistent with RPA recognition of DNA-damage playing a critical role in NER.

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