To determine the consequences of defective proofreading in mammals, mice with a point mutation in the exonuclease-encoding domain (D400A corresponding to yeast D407A) of DNA polymerase-delta (pol-delta) were created (Pold1D400A/D400A). Pol-delta is a primary replicative enzyme in eukaryotes. Its catalytic subunit contains a proofreading active site formed by the conserved motifs Exo I, Exo II and Exo III that include four invariant carboxylate residues thought to be essential for catalysis. To determine the phenotype of the mutant allele, a chimeric male was crossed with C57BL/6J females and the resultant heterozygous F1 mice were interbred to generate a cohort of F2 animals. The mice were evaluated and had exhibited normal development and weight gain. Together with embryo viability unaffected by the mutant allele and subsequent breeding experiments showing both male and female Pold1D400A/D400A mice remaining fertile, pol-delta proofreading was shown to not be required for mouse reproduction or development. Beyond two months of age, Pold1D400A/D400A mice began to develop cancer and die. 43% of the Pold1D400A/D400A mice and none of the Pold1+/D400A or wild-type mice had developed tumors 12 months into the study. The tumors were derived from two different cell lineages. 70% of the tumors originated from mesenchymal tissue and 30% from epithelial tissue. Surviving Pold1D400A/D400A mice continue to develop early signs of tail skin lesions (cracking, small nodules) and are being monitored for further tumor progression and metastatic disease.
The findings above reveal that pol-delta proofreading reduces spontaneous tumor development in mice, however, the role of pol-delta variants in human cancers remains to be defined.
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