DNA polymerases alpha and gamma during pre-emergence and early larval development of Artemia.

Abstract

DNA polymerases alpha and gamma have been studied in cryptobiotic cysts and developing embryos and larvae of the brine shrimp Artemia. The two enzymes readily separate on Cibacron blue 3-GA Matrex gel. Assay requirements with activated DNA as primer-template are pH 8.0, 1 mM Mg2+, 50 mM K+ for DNA polymerase alpha and pH 8.4, 10 mM Mg2+, 80 mM K+ for DNA polymerase gamma. DNA polymerase alpha is inhibited by N-ethylmaleimide (94% and 100% at 1 mM and 10 mM respectively) and aphidicolin (96% at 60 microM). DNA polymerase gamma is also sensitive to N-ethylmaleimide (83% and 100% inhibition at 1 mM and 10 mM respectively) but is resistant to aphidicolin. 2',3'-Dideoxythymidine 5'-triphosphate (ddTTP) inhibits the gamma polymerase by 88% when in fivefold excess over dTTP whereas the alpha polymerase is unaffected by this compound. DNA polymerase alpha has a sedimentation coefficient of 7.6 S which is reduced to 6.2 S by a phenylmethylsulphonyl fluoride-sensitive proteinase. The gamma polymerase sediments at 8.3 S. No DNA polymerase beta activity could be detected. After the reinitiation of development both activities increased twofold up to 8 h (gamma polymerase) and 16 h (alpha polymerase), then declined before the onset of nuclear DNA replication after hatching. Thymidine kinase activity increased over 200-fold up to the time of replication. Analysis on Percoll density gradients of the intracellular distribution of both polymerases during development suggests that the changes in their activities may be due to migration from storage sites to replication complexes in the nuclei and mitochondria. The content of the mitochondrial DNA polymerase gamma in different batches of cysts may reflect the relative viabilities of the cysts.