GENOME WIDE SPONTANEOUS MUTATION IN HUMAN-CELLS DETERMINED BY THE SPECTRUM OF MUTATIONS IN HPRT CDNA GENES

We have studied spontaneous mutagenesis in rive hprt cDNA genes integr ated at five different genomic positions in a human lymphoblastoid cel l line (TK6). The spectra of 40 mutants from each position were combin ed to obtain a mutation spectrum of the overall genome. This collectio n of mutants was used to assess the contribution of several mutagenic processes to spontaneous mutagenesis. Deletions and single base pair c hanges account for the majority of the mutants and arise in approximat ely equal amounts (43 and 41%, respectively). The majority of the dele tions and insertions are < 5 bp and are likely to be caused by templat e-directed misalignment (slippage) during replication. To account for frameshifts at non-iterated sites we propose a slightly different temp late-directed replication error model. A considerable amount of the ob served base pair changes can also be explained by this last model, but several other processes leading to base pair changes such as depurina tion, deamination or spontaneously arising DNA damage are likely to co ntribute as well. We have compared this spectrum with mutation spectra in the endogenous hprt genes using published mutation data. It is sho wn that in the endogenous genes the contribution of base pair substitu tions is much larger (71%) than in the hprt cDNA integrates and that d eletions are less frequently observed (20%). The mutation rates of the integrated hprt cDNA genes show a mean increase of 30-fold as compare d with the endogenous hprt gene. This results in a 60-fold increase of the absolute rate of deletion in the hprt cDNA genes and in a 15-fold increase of the base pair substitution rate. Replication errors such as slippage or the mechanism proposed in this study probably account t o a large extent for this increase.