Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair

EMBO J. 2001 Jul 2;20(13):3554-64. doi: 10.1093/emboj/20.13.3554.

Abstract

The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system. Although XPA-RPA has been implicated in damage recognition, its activity in the DNA repair pathway remains controversial. By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak preference of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations. Further screening with artificially distorted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA. Conversely, RPA recognizes single-stranded sites but not backbone bending. Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA. The affinity of XPA for sharply bent duplexes, characteristic of architectural proteins, is not compatible with a direct function during recognition of nucleotide lesions. Instead, XPA in conjunction with RPA may constitute a regulatory factor that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pair Mismatch
  • Base Sequence
  • Binding Sites
  • DNA / chemistry*
  • DNA / metabolism
  • DNA Helicases / metabolism
  • DNA Repair / physiology*
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli
  • Humans
  • Hydrogen Bonding
  • Kinetics
  • Nucleic Acid Conformation
  • Nucleic Acid Heteroduplexes / chemistry
  • Nucleic Acid Heteroduplexes / metabolism
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • RNA-Binding Proteins / isolation & purification
  • RNA-Binding Proteins / metabolism*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Replication Protein A
  • Xeroderma Pigmentosum Group A Protein

Substances

  • DNA-Binding Proteins
  • Nucleic Acid Heteroduplexes
  • Oligodeoxyribonucleotides
  • RNA-Binding Proteins
  • RPA1 protein, human
  • Recombinant Proteins
  • Replication Protein A
  • XPA protein, human
  • Xeroderma Pigmentosum Group A Protein
  • DNA
  • DNA Helicases