Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells.
We used a shuttle vector based on the Epstein-Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells. Plasmid DNA was incubated at pH 2 at 40 degrees C for various times to induce up to 20 apurinic (AP) sites per 9.7-kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient. After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid-encoded herpes simplex virus type 1-thymidine kinase gene. Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells. The mutant frequency at 20 AP sites/plasmid was 10-fold to 13-fold greater than that observed for untreated DNA. Deletion and frameshift events accounted for 46-55% of the mutants induced by depurination. The induced deletions were relatively small (median size, 100-150 bp) and characterized by short (1-5 bp) regions of sequence homology at the endpoints. These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP-site-induced template dislocation during DNA synthesis. A broad spectrum of base-substitution mutations, which accounted for 19-36% of the induced mutants, was observed. The apparent preference for insertion opposite AP sites in human cells was G (43-55%) greater than A approximately C (18-21%) greater than T (9-14%). Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.
Klinedinst
D K
DK
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Drinkwater
N R
NR
D001080:Apurinic Acid / Q000235:genetics / Q000378:metabolism; D001260:Ataxia Telangiectasia / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003198:Computer Simulation; D004926:Escherichia coli; D005260:Female; D016368:Frameshift Mutation; D005822:Genetic Vectors; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013014:SOS Response, Genetics; D012689:Sequence Homology, Nucleic Acid; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism
D001080:Apurinic Acid; D013937:Thymidine Kinase