Dataset Information

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.

SUBMITTER: Frank EG

PROVIDER: S-EPMC5303534 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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

Frank Ekaterina G EG McDonald John P JP Yang Wei W Woodgate Roger R

DNA repair 20161221

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 synth ...[more]

PMID: 28077247

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Project description:Pseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor RhlR, an interaction that enhances the affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, the virulence function of PqsE can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing enhanced green fluorescent protein-PqsE fusions to define the protein interactomes of wild-type PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wild-type PqsE while not forming associations with PqsE(D73A). In the ΔrhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase for RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing. IMPORTANCE Pseudomonas aeruginosa causes hospital-borne infections in vulnerable patients, including immunocompromised individuals, burn victims, and cancer patients undergoing chemotherapy. There are no effective treatments for P. aeruginosa infections, which are usually broadly resistant to antibiotics. Animal models show that, to establish infection and to cause illness, P. aeruginosa relies on an interaction between two proteins, namely, PqsE and RhlR. There could be additional protein-protein interactions involving PqsE, which, if defined, could be exploited for the design of new therapeutic strategies to combat P. aeruginosa. Here, we reveal previously unknown protein interactions in which PqsE participates, which will be investigated for potential roles in pathogenesis.

Dataset Information

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

Publications

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

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