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

ABSTRACT: DNA polymerase ? (pol ?) 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 ? 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 ?. Four-subunit human pol ? variants that harbor or lack 3' ? 5'-exonucleolytic proofreading activity were purified from Escherichia coli. The pol ? L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol ?. pol ? L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol ? L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol ?. However, pol ? 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.

SUBMITTER: Schmitt MW 

PROVIDER: S-EPMC2952227 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature