Related Experiment Videos
Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1
W H Koch1, A R Fernández de Henestrosa, R Woodgate
1Molecular Biology Branch, Food and Drug Administration, 20204, Washington, DC, USA.
Abstract:
Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an approximately 7kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB((R394)) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a DeltaumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.
Insights
Researchers developed a new assay to identify umuC-like genes in bacteria. This method identified mucAB-like operons on plasmids, revealing inactive MucB proteins but also potential new DNA polymerases.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Lesion-replicating DNA polymerases are crucial for DNA repair and mutagenesis.
- The UmuC-like subfamily of these polymerases can be further classified into five groups, including MucAB.
- Some UmuC-like genes are inactive in their native state but can enhance mutagenesis when overexpressed.
Purpose of the Study:
- To develop a functional assay for identifying umuC-like genes in situ.
- To investigate the presence and activity of umuC-like genes on bacterial plasmids R394 and Rts-1.
Main Methods:
- Phylogenetic analysis to classify DNA polymerases.
- Design of degenerate primers targeting conserved regions of UmuC-like polymerases.
- DNA sequencing of plasmid regions and analysis of gene expression.
- Complementation assays using an Escherichia coli strain deficient in umuDC genes.
Main Results:
- Degenerate primers successfully identified mucAB-like operons on IncT plasmids R394 and Rts-1.
- Sequence analysis revealed LexA-regulated genes similar to E. coli tus and impC adjacent to mucAB((R394)).
- Both R394 and Rts-1 mucB genes contained insertions leading to truncated, inactive MucB proteins.
- Plasmid R394 did not restore mutagenesis, but Rts-1 significantly increased MMS-induced SOS mutagenesis.
Conclusions:
- The developed assay is effective for identifying umuC-like genes in situ.
- The Rts-1 plasmid may encode an uncharacterized umu-like DNA polymerase responsible for enhanced mutagenesis.
- Further investigation is needed to identify the novel umu-like homolog on the Rts-1 plasmid.