129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

Abstract

cMice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Pol) gene and are therefore considered Pol deficient. When we amplified Pol mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Pol isoform lacking exon 2. Pol mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Pol protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Pol, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Pol ؊/؊ embryonic fibroblasts derived from a new, fully deficient Pol knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Pol in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Pol, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Pol activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.

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

“…As reported by others, the exon-2-less derivative is unstable [16, 17], presumably because its structural integrity is severely compromised (Fig 1.). While we were unable to purify sufficient quantities of the mutant protein from E.coli extracts, we were able to purify the exon-2-less polι mutant as a GST-fusion protein from baculovirus infected insect cells (Fig.…”

“…The tertiary structure of polι without the 42 residues encoded by exon 2 is therefore highly likely to be partially unfolded. Given this scenario, it is hardly surprising that the mutant protein is incredibly unstable in vivo and is rapidly degraded by the proteasome in human cells [16]. …”

“…Because of this fact, it has been hypothesized that the exon-2-less isoform may retain significant biological activity in vitro and in vivo . Indeed, a recent study by Aoufouchi et al ., appears to support this notion [16]. …”

“…However, the assumption that 129-derived strains of mice are truly deficient in polι activity has recently been questioned by the fact that in 129-derived strains, exon-2 is skipped during mRNA maturation [15, 16]. The extent of the exon skipping appears to be dependent upon the precise 129-derived strain analyzed, with exon-2 skipping in 129X1/SvJ significantly lower than that observed in 129/Ola mice [16].…”

“…The extent of the exon skipping appears to be dependent upon the precise 129-derived strain analyzed, with exon-2 skipping in 129X1/SvJ significantly lower than that observed in 129/Ola mice [16]. The extent of exon-2 skipping also appears to be tissue specific [17].…”