N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation.
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.
Feng
Zhaohui
Z
Department of Environmental Medicine, New York University Medical Center, Tuxedo, New York 10987, USA.
Hu
Wenwei
W
Rom
William N
WN
Beland
Frederick A
FA
Tang
Moon-shong
MS
D000611:Aminobiphenyl Compounds / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002273:Carcinogens / Q000737:chemistry / Q000378:metabolism*; D018899:CpG Islands; D003596:Cytosine / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D019175:DNA Methylation; D004706:Endodeoxyribonucleases / Q000737:chemistry; D029968:Escherichia coli Proteins; D005091:Exons / Q000235:genetics; D016158:Genes, p53 / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism*; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D001749:Urinary Bladder Neoplasms / Q000209:etiology / Q000378:metabolism
D000611:Aminobiphenyl Compounds; D002273:Carcinogens; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D009153:Mutagens; C014593:N-hydroxy-4-aminobiphenyl; D003596:Cytosine; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC