Organism ensure the fidelity of their DNA by three overlapping pathways namely base selection, proofreading and mismatch repair. These pathways cumulate in replication fidelity of ~10^-10 mutations per bp per generation in E. coli. By modulating the expression or activity of different genes participating in these fidelity mechanisms respectively expressing dominant negative variants the mutation rate can be increased substantial. Recently, mutation rates up to 6.2×10^-6 mutations per bp per generation were obtained by using a plasmid-borne mutator system. (Badran, Liu 2015) We aim to make this mutator plasmid available for the iGEM community by rebuilding it with defined iGEM parts (see our BBa_K208117). We envision an application in the directed evolution of our binding protein. Furthermore implementation in other directed evolution approaches could be feasibly in which our plasmid pose an alternative to error-prone PCR (Wilson, Keefe 2001) or exogene mutagens (reviewed in Steensels et al. 2014; Wong et al. 2006).

DNA is the single blueprint for all proteins of an organism. Minor changes in the DNA sequence are mostly without significant consequences, but in some cases the consequences are detrimental e.g. by rendering encoded crucial proteins useless. Therefore, an effective system of DNA repair mechanisms emerged during evolution. The DNA fidelity system is composed of three main mechanisms, which contribute to the high fidelity. The first fidelity mechanism lies in selecting the correct base for incorporation during replication of DNA. This process of base selection reduces the error rate by 10⁵ mutations per bp per generation. During replication, the acting polymerase (mainly polymerase III in E. coli) directly corrects falsely incorporated bases. This process is called proofreading and reduces the error rate by 10² mutations/bp/generation. Other mutations in the DNA e.g. created through replication errors or other mutagenic sources are repaired by the mismatch repair systems. This system reduces the error rate by 10³ mutations per bp per generation. (Schaaper 1993; Fijalkowska et al. 2012) All these fidelity mechanisms contributed to the error rate reduction to about 10^-10 mutations per bp per generation. (Lee et al. 2012).
Many variants of the participating proteins with (partial) loss-of-function were identified and described in the literature (Schaaper, Radman 1989; Degnen, Cox 1974; Junop et al. 2003; Fowler, Schaaper 1997; Nghiem et al. 1988). The common phenotype upon the mutants was a strongly increased mutation rate. It did not take long before better-characterised hypermutator strains arised as a tool to study the effect of random mutations on proteins or to improve proteins trough means of directed evolution (Greener et al. 1997; Selifonova et al. 2001; Stefan et al. 2001). A common issue among different applications of genomic mutator genes is the general genetic instability and frequent unviability of these strains. For example, the E. coli strain, which chromosomally harbors the strongest known mutator dnaQ926 is almost completely unviable without supporting mutations (Fijalkowska, Schaaper 1996)). Therefore, recent development was directed to the rational design of a plasmid-borne mutator system. By combining the strongest mutator capabilities with a tight inducible promoter should result in a controllable mutagenesis system, generally circumventing many issues of chromosome-based hypermutator strains. Furthermore, the rational combination of different groups of mutator genes enables combinations, which have a very balanced mutagenesis spectra
Our mutation system was designed to use an untargeted mutation approach as an alternative to the error-prone polymerase I. Therefore, we tried to reconstruct the previously described mutator plasmid (Badran, Liu 2015), which consists of six different gene.

Overexpression of these mutator genes rises the mutation frequency in E. coli to 6.2×10^-6 per bp per generation with induction and only ~4×10^-9 per bp per generation without induction, respectively with a diverese mutation spectrum.

Tabelle mit dem Mutationsspektrum

The dnaQ gene encodes ε-subunit of E. coli DNA polymerase III, which is responsible for the proof reading activity of this enzyme. DnaQ confers 3'-exonuclease activity, which excises falsely incorporated bases during replication. DnaQ926 contains two amino acid substitutions (D12A, E14A) in the Exo I region, which prevents coordination of an essential metal ion and therefore abolishes the proofreading activity(Fijalkowska, Schaaper 1996). The overall structure of DnaQ and interactions with other proteins of the DNA polymerase III complex are not affected by these substitutions. dnaQ926 acts as a dominant negative gene causing a strong mutator phenotype even in E. coli strains with chromosomal wild type dnaQ (Cox, Horner 1986). DnaQ926 competes against DnaQ about incorporation into the DNA polymerase III complex. Incorporation of DnaQ926 leads to proofreading deficient replication complexes. Therefore, overexpression or plasmid-borne expression of dnaQ926 is supposed to yield a increased mutation effect. Another side effect of dnaQ926 is, that the E. coli mismatch repair system is overwhelmed by the large amount of errors introduced by the defective polymerase III complexes. The saturation of this repair mechanism greatly decreases it’s effectivity and prevents repair of various mutations. (Schaaper, Radman 1989; Damagnez et al. 1989)
The exact rate and mutagenesis spectrum of DnaQ926 differs between the used media. Rich media yield high mutation frequency (3.7×10⁴ over background) with mostly transitions, while minimal media cause less mutations (4.8×10² over background) and mostly transversions (mainly AT → TA). The faster growth of bacteria in rich media decreases the time the time to remove errors before cell division via the repair mechanisms. (Schaaper 1988)

coming soon

As mentioned before the main factor of replication fidelity is the correct base incorporation during replication. This is mainly dependent on the α-subunit of DNA polymerase III, but the intracellular concentrations of the single nucleotidetriphosphates are important too (ref?). A strong imbalance between the single nucleotide-triphosphates or a high concentration of base analogs can increases the rate of incorporation of a wrong base (Gon et al. 2011).
One possible way to achieve this imbalance is the overexpression of emrR. EmrR is a transcriptional repressor of the emrAB operon. This operon encodes an efflux pump, which is linked to multidrug resistance (Lomovskaya et al. 1995). However, the native function of EmrAB is the export of intermediates of the purine and pyrimidine biosynthesis and metabolism. An overexpression of emrR reduces the concentration of EmrA and EmrB leading to increased concentration of nucleotide precursors inside the cell. At these higher concentrations, these nucleotide precursors can act as base analogs. Mutations are caused either by the incorporated nucleotide precursor or via error prone repair mechanisms.(Gabrovsky et al. 2005)

Cytidin deaminases (cda) are DNA editing enzymes in eukaryotes, which have multiple functions like the generation of antibody diversity, defence against retroviruses and demethylation during development (Lada et al. 2011).
The main activity of Cdas is the deamination from cytidin to uracil in CG base pairs. The resulting UG base pairs split in replication in one CG and one UA basepair. Therefore, the main mutations created by Cdas are C → T transitions.

Organisms have created mechanism to repair mismatches as UG pairs via the base excision repair (BER). Thereby the mismatched base is cut out by the uracil-DNA-glycosylase (Ung). The resulting abasic site (AP-site) is cleaved by an AP-endonuclease and the patch is filled by a variety of specialised proteins (Krokan, Bjoras 2013).
Overexpression of eukaryotic cytidin deaminase increases the mutation rate in bacteria. The Cda1 of Pteromyzon marinus was identified in a comprehensive screen as the most potent cytidin deaminase when expressed in E. coli. Overexpression of P. marinus cda1 increases the mutation rate to 10^-6-10-5 /bp. In ung deficient strains the effect was amplified by a factor of 10^? due to the lack of UNG induced BER (Lada et al. 2011).
The CDS of P. marinus cda1 was codon optimized for E. coli integrated into our mutagenesis plasmid to increase the expression. Moreover, the uracil-DNA-glycosylase inhibitor (ugi) was added. This short protein mimics the structure of abasic sides, binds to UNG and inhibits the UNG mediated BER pathway.