TY - JOUR
T1 - Mutagenesis by human immunodeficiency virus reverse transcriptase
T2 - Incorporation of O6-methyldeoxyguanosine triphosphate
AU - Hizi, Amnon
AU - Kamath-Loeb, Ashwini S.
AU - Rose, Karl D.
AU - Loeb, Lawrence A.
N1 - Funding Information:
This work was supported by funding from National Institutes of Allergy and Infectious Diseases grant AI38180 to LAL. We thank S. Hughes (National Cancer Institute, Frederick, MD) for generously supplying purified HIV-1 reverse transcriptase, S. Mitra (University of Texas Medical Branch, Galveston, TX) for providing bacterial MGMT, M.S. Solomon and J. Essigmann (Massachusetts Institute of Technology, Cambridge, MA) for synthesizing O 6 -methyl-dGTP, and J. Silber (University of Washington, Seattle, WA) for providing O 6 -[ 3 H]methylguanine containing DNA.
PY - 1997/3/4
Y1 - 1997/3/4
N2 - The high frequency of incorporation of non-complementary nucleotides by HIV-1 reverse transcriptase is likely to be a major factor in the exceptionally rapid accumulation of viral mutations during the course of AIDS infections, To investigate whether this high level of infidelity is also associated with the incorporation of nucleotide analogs, we analyzed O6-methyldeoxyguanosine triphosphate and compared the incorporation of this analog by HIV-1 reverse transcriptase to that catalyzed by other DNA synthesizing enzymes. Our results indicate that O6-methyldeoxyguanosine triphosphate serves as a substrate for DNA synthesized in vitro by HIV-1 RT on both DNA and RNA templates. The product DNA contains the modified purine; it is sensitive to the repair enzyme, O6-methylguanine methyltransferase, which specifically reacts with DNA containing methylated guanines at the O6 position, Using a forward mutation assay we demonstrated that the nucleotide analog incorporated by HIV-1 RT is mutagenic. The mutations produced are single-base substitutions opposite template thymidines and result in A:T → G:C transitions. The incorporation of a mutagenic nucleotide by HIV-1 RT highlights the possibility of increasing the rate of mutagenesis of HIV by the use of nucleotides that form non-complementary base pairs at high frequency.
AB - The high frequency of incorporation of non-complementary nucleotides by HIV-1 reverse transcriptase is likely to be a major factor in the exceptionally rapid accumulation of viral mutations during the course of AIDS infections, To investigate whether this high level of infidelity is also associated with the incorporation of nucleotide analogs, we analyzed O6-methyldeoxyguanosine triphosphate and compared the incorporation of this analog by HIV-1 reverse transcriptase to that catalyzed by other DNA synthesizing enzymes. Our results indicate that O6-methyldeoxyguanosine triphosphate serves as a substrate for DNA synthesized in vitro by HIV-1 RT on both DNA and RNA templates. The product DNA contains the modified purine; it is sensitive to the repair enzyme, O6-methylguanine methyltransferase, which specifically reacts with DNA containing methylated guanines at the O6 position, Using a forward mutation assay we demonstrated that the nucleotide analog incorporated by HIV-1 RT is mutagenic. The mutations produced are single-base substitutions opposite template thymidines and result in A:T → G:C transitions. The incorporation of a mutagenic nucleotide by HIV-1 RT highlights the possibility of increasing the rate of mutagenesis of HIV by the use of nucleotides that form non-complementary base pairs at high frequency.
KW - HIV reverse transcriptase
KW - Lethal mutagenesis
KW - Mutagenesis
KW - Nucleotide analog
KW - O-Methyldeoxyguanosine
UR - http://www.scopus.com/inward/record.url?scp=0031048416&partnerID=8YFLogxK
U2 - 10.1016/S0027-5107(96)00217-5
DO - 10.1016/S0027-5107(96)00217-5
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AN - SCOPUS:0031048416
SN - 0027-5107
VL - 374
SP - 41
EP - 50
JO - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
JF - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
IS - 1
ER -