The frequency of CC to TT tandem mutations in mismatch repair-deficient cells is increased in a cytosine run

Mononucleotide runs are hot spots for frameshift mutations in mismatch repair (MMR)-deficient cells. However, a role for mononucleotide runs in the formation of base pair substitutions has not been tested. Previously, we demonstrated that ultraviolet radiation C (UVC)- or reactive oxygen species-induced CC to TT tandem mutations are markedly enhanced in MMR-deficient cells. The target for the mutational analysis was two cytosines in a run of five cytosines (5C) within mouse Aprt. Because mutation from C to T for either or both of the two critical cytosines created a codon yielding a functional Aprt protein, this assay allowed both single and tandem substitutions to be quantified and the relative ratios compared. To determine if the cytosine run increased the frequency of single and/or tandem base pair substitutions, alternative constructs were created in which the cytosine run was disrupted by flanking the target cytosines with either thymines (2Cpyr) or adenines (2Cpur). Disruption of the cytosine run dramatically decreased the frequency of UVC-induced tandem mutations in the 2Cpyr and 2Cpur constructs, as compared with the 5C construct. Moreover, CC to TT tandem mutations occurred spontaneously or were induced by oxidative stress only within the 5C construct. These results demonstrate that CC to TT tandem mutations in MMR-deficient cells form more readily in a homocytosine run than in a sequence limited to two cytosines.