The steric gate amino acid tyrosine 112 is required for efficient mismatched-primer extension by human DNA polymerase kappa.

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, which distinguishes dNTPs from rNTPs by sensing the 2'-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.

Mutagenic specificity of 2-acetylaminonaphthalene-derived DNA adduct in mammalian cells.

2-Acetylaminonaphthalene (2-AAN) has been recognized as a urinary bladder carcinogen in humans. The deacetylated form, 2-aminonaphthalene (2-AN), is metabolized in vivo and reacts primarily with guanine residues in DNA, resulting in the formation of dG-N(2)-aminonaphthalene (dG-N(2)-AN) adduct. Phosphoramidite chemical procedure has recently been established in our laboratory to prepare oligodeoxynucleotides containing a single dG-N(2)-acetylaminonaphthalene (dG-N(2)-AAN) adduct. Oligodeoxynucleotides ((5')TCCTCCTNXCCTCTC, where X is dG or dG-N(2)-AAN and N is C, A, T or G) with different bases 5' flanking to the lesion were prepared and were inserted into a single-strand shuttle vectors and used to establish the mutational frequency and specificity of dG-N(2)-AAN adduct in simian kidney cells. dG-N(2)-AAN adduct promoted preferential incorporation of dCMP, the correct base, opposite the lesion. When the 5' flanking base to the lesion was C, A or T, the mutational frequency was under 2.1%. When G flanked to the lesion, the mutational frequency was slightly increased to 4.2%. Misincorporation of dAMP, dTMP, and/or dGMP varied depending on the 5' flanking base. When dG-N(2)-AAN was positioned at codon 61 of noncoding strand of human c-Ha-ras1 gene ((5')TCCTCCTXGCCTCTC, where X is dG-N(2)-AAN), the mutational frequency was 6.7%; G-->T transversions (4.7%), followed by G-->A transition (2.0%), were observed. These results demonstrated that dG-N(2)-AAN is a weak mutagenic lesion in mammalian cells. The influence of 5' flanking sequence context was observed on the mutational frequency and specificity of this adduct.

Mutagenic potential of benzo[a]pyrene-derived DNA adducts positioned in codon 273 of the human P53 gene.

Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo[a]pyrene. We incorporated a single (+)- or (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells. Oligodeoxyribonucleotides ((5)(')GAGGTGCG(BPDE)TGTTTGT) modified with (+)- or (-)-trans-dG-N(2)-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells. Progeny plasmids were then used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base. We determined the mutational frequencies associated with (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE adduction to be 26.5% and 17.5%, respectively. The predominant mutations generated by both stereoisomers were G --> T transversions, with some G --> A transitions. When the cytosine 5' to dG-N(2)-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged. Thus, the mutational "hot spot" at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE-DNA adducts positioned at this site.