Active site mutations in mammalian DNA polymerase delta alter accuracy and replication fork progression.

DNA polymerase delta (pol delta) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol delta L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol delta. Four-subunit human pol delta variants that harbor or lack 3' --> 5'-exonucleolytic proofreading activity were purified from Escherichia coli. The pol delta L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol delta. pol delta L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol delta L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol delta. However, pol delta L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression.