Mouse DNA polymerase ι lacking the forty-two amino acids encoded by exon-2 is catalytically inactive in vitro.

In 2003, we reported that 129-derived strains of mice carry a naturally occurring nonsense mutation at codon 27 of the Poli gene that would produce a polι peptide of just 26 amino acids, rather then the full-length 717 amino acid wild-type polymerase. In support of the genomic analysis, no polι protein was detected in testes extracts from 129X1/SvJmice, where wild-type polι is normally highly expressed. The early truncation in polι occurs before any structural domains of the polymerase are synthesized and as a consequence, we reasoned that 129-derived strains of mice should be considered as functionally defective in polι activity. However, it has recently been reported that during the maturation of the Poli mRNA in 129-derived strains, exon- 2 is sometimes skipped and that an exon-2-less polι protein of 675 amino acids is synthesized that retains catalytic activity in vitro and in vivo. From a structural perspective, we found this idea untenable, given that the amino acids encoded by exon-2 include residues critical for the coordination of the metal ions required for catalysis, as well as the structural integrity of the DNA polymerase. To determine if the exon-2-less polι isoform possesses catalytic activity in vitro, we have purified a glutathione-tagged full-length exon-2-less (675 amino acid) polι protein from baculovirus infected insect cells and compared the activity of the isoform to full-length (717 amino acid) GST-tagged wild-type mouse polι in vitro. Reaction conditions were performed under a range of magnesium or manganese concentrations, as well as different template sequence contexts. Wild-type mouse polι exhibited robust characteristic properties previously associated with human polι's biochemical properties. However, we did not detect any polymerase activity associated with the exon-2-less polι enzyme under the same reaction conditions and conclude that exon-2-less polι is indeed rendered catalytically inactive in vitro. Published by Elsevier B.V.