Preferential binding of human full-length XPA and the minimal DNA binding domain (XPA-MF122) with the mitomycin C-DNA interstrand cross-link

Biochemistry. 2001 Jun 19;40(24):7158-64. doi: 10.1021/bi002820u.

Abstract

Nucleotide excision repair (NER) is an important cellular mechanism that removes radiation-induced and chemically induced damage from DNA. The XPA protein is involved in the damage recognition step of NER and appears to function by binding damaged DNA and recruiting other proteins to the site. It may also play a role in subsequent steps of NER through interaction with other repair proteins. Interstrand cross-links are of particular interest, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery. Using 14 and 25 bp duplex oligonucleotides containing a defined, well-characterized single mitomycin C (MMC)-DNA interstrand cross-link, we have shown through gel shift analysis that both XPA and a minimal DNA binding domain of XPA (XPA-MF122) preferentially bind to MMC-cross-linked DNA with a greater specificity and a higher affinity (>2-fold) than to the same undamaged DNA sequence. This preferential binding to MMC-cross-linked DNA occurs in the absence of other proteins from the NER complex. Differences in binding affinity and specificity were observed among the different protein-DNA combinations that were both protein and DNA specific. Defining XPA-MMC-DNA interactions may aid in elucidating the mechanism by which DNA cross-links and other forms of DNA damage are recognized and repaired by the NER machinery in eukaryotic cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding, Competitive
  • Cross-Linking Reagents / metabolism*
  • DNA / metabolism*
  • DNA Repair*
  • DNA-Binding Proteins / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Mitomycin / metabolism*
  • Nucleic Acid Heteroduplexes / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA-Binding Proteins / metabolism*
  • Xeroderma Pigmentosum Group A Protein

Substances

  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • Nucleic Acid Heteroduplexes
  • RNA-Binding Proteins
  • XPA protein, human
  • Xeroderma Pigmentosum Group A Protein
  • Mitomycin
  • DNA