Interactions of human replication protein A with single-stranded DNA adducts

Yiyong Liu, Zhengguan Yang, Christopher D. Utzat, Yu Liu, Nicholas E. Geacintov, Ashis K. Basu, Yue Zou

Research output: Contribution to journalArticlepeer-review

Abstract

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-amino-pyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA. the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8-9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

Original languageEnglish (US)
Pages (from-to)519-526
Number of pages8
JournalBiochemical Journal
Volume385
Issue number2
DOIs
StatePublished - Jan 15 2005

Keywords

  • Adduct
  • Binding affinity
  • DNA damage recognition
  • Fluorescence spectroscopy
  • Human replication protein A
  • Single-stranded DNA

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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