title: Mouse DNA polymerase ι lacking the forty-two amino acids encoded by exon-2 is catalytically inactive in vitro.
abstract: 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.
authors.affiliation: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
authors.forename: Ekaterina G
authors.initials: EG
authors.lastname: Frank
authors.affiliation: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: McDonald
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.affiliation: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA. Electronic address: woodgate@nih.gov.
authors.forename: Roger
authors.initials: R
authors.lastname: Woodgate
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D018389:Codon, Nonsense; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D051379:Mice; D011487:Protein Conformation; D016415:Sequence Alignment; D017384:Sequence Deletion; D000094769:DNA Polymerase iota
chemical_list: D018389:Codon, Nonsense; D004259:DNA-Directed DNA Polymerase; D000094769:DNA Polymerase iota
keywords: 129-derived strains of mice; DNA polymerase ι; Mutagenesis; Translesion DNA synthesis; Y-family DNA polymerase