Mismatch repair deficient human cells: spontaneous and MNNG-induced mutational spectra in the HPRT gene

We have determined both the spontaneous and N-methyl-N'-nitro-N-nitrosoguan idine (MNNG)-induced mutational spectra in the HPRT gene of human cells (MT 1) defective in the mismatch repair gene hMSH6 (GTBP). Eight of nine exons and nine of sixteen intronic flanking sequences were scanned, encompassing > 900 bp of the HPRT gene. Mutant hotspots were detected and separated by d ifferences in their melting temperatures using constant denaturant capillar y electrophoresis (CDCE) or denaturing gradient gel electrophoresis (DGGE). A key finding of this work is that a high proportion of all HPRT inactivati ng mutations is represented by a small number of hotspots distributed over the exons and mRNA splice sites. Thirteen spontaneous hotspots and sixteen MNNG-induced hotspots accounted for 55% and 48% of all 6TG(R) point mutatio ns, respectively. MNNG-induced hotspots were predominantly G:C --> A:T tran sitions. The spontaneous spectrum of cells deficient in hMSH6 contained tra nsversions (A:T --> T:A, G:C --> T:A, A:T --> C:G), transitions (A:T --> G: C), a plus-one insertion, and a minus-one deletion. Curiously, G:C --> A:T transitions, which dominate human germinal and somatic point mutations were absent from the spontaneous hMSH6 spectra. (C) 2000 Elsevier Science B.V. All rights reserved.