N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation

Biochemistry. 2002 May 21;41(20):6414-21. doi: 10.1021/bi020093s.


4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer. Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis. Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features. To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level. We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP. Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding. We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP. We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated. These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer.

Publication types

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

MeSH terms

  • Aminobiphenyl Compounds / chemistry
  • Aminobiphenyl Compounds / metabolism*
  • Base Sequence
  • Binding Sites / genetics
  • Carcinogens / chemistry
  • Carcinogens / metabolism*
  • CpG Islands
  • Cytosine / metabolism*
  • DNA Adducts / chemistry
  • DNA Adducts / metabolism*
  • DNA Damage
  • DNA Methylation*
  • Endodeoxyribonucleases / chemistry
  • Escherichia coli Proteins*
  • Exons / genetics
  • Genes, p53* / drug effects
  • Humans
  • Molecular Sequence Data
  • Mutagens / chemistry
  • Mutagens / metabolism*
  • Nucleic Acid Denaturation / drug effects
  • Urinary Bladder Neoplasms / etiology
  • Urinary Bladder Neoplasms / metabolism


  • Aminobiphenyl Compounds
  • Carcinogens
  • DNA Adducts
  • Escherichia coli Proteins
  • Mutagens
  • N-hydroxy-4-aminobiphenyl
  • Cytosine
  • Endodeoxyribonucleases
  • endodeoxyribonuclease uvrABC