| First Author | Frank EG | Year | 2017 |
| Journal | DNA Repair (Amst) | Volume | 50 |
| Pages | 71-76 | PubMed ID | 28077247 |
| Mgi Jnum | J:349543 | Mgi Id | MGI:6391989 |
| Doi | 10.1016/j.dnarep.2016.12.004 | Citation | Frank EG, et al. (2017) Mouse DNA polymerase iota lacking the forty-two amino acids encoded by exon-2 is catalytically inactive in vitro. DNA Repair (Amst) 50:71-76 |
| abstractText | 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 poliota peptide of just 26 amino acids, rather then the full-length 717 amino acid wild-type polymerase. In support of the genomic analysis, no poliota protein was detected in testes extracts from 129X1/SvJmice, where wild-type poliota is normally highly expressed. The early truncation in poliota 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 poliota 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 poliota 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 poliota isoform possesses catalytic activity in vitro, we have purified a glutathione-tagged full-length exon-2-less (675 amino acid) poliota protein from baculovirus infected insect cells and compared the activity of the isoform to full-length (717 amino acid) GST-tagged wild-type mouse poliota in vitro. Reaction conditions were performed under a range of magnesium or manganese concentrations, as well as different template sequence contexts. Wild-type mouse poliota exhibited robust characteristic properties previously associated with human poliota''s biochemical properties. However, we did not detect any polymerase activity associated with the exon-2-less poliota enzyme under the same reaction conditions and conclude that exon-2-less poliota is indeed rendered catalytically inactive in vitro. |
