Reporter gene fusions with proteins involved in DNA repair are a common technique to investigate the DNA repair machinery (Kidane et al., 2004; Sanchez et al., 2006). It sounds quite logical to investigate DNA repair machineries for nucleotide or base excision repair, HR, or other repair pathways using reporter gene fusions, but a major problem is to practically target a cell having a DNA lesion and needing a certain repair pathway. To circumvent this problem DNA damaging agents as mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) or methyl methane sulfonate (MMS) are often used leading to stalling or collapse of the replication fork (Sanchez et al., 2006). MMC even results in DSB.
Other systems to investigate DSB repair are the arabinose inducible I-Sce-I endonuclease or the xylose inducible HO endonuclease system form Saccharomyces cerevisiae. Both systems can be used to generate DSBs at certain cut sites (Haber, 2002; Kidane and Graumann, 2005; Lesterlin et al., 2014). However, the investigation of natural occurring DNA damages during replication or transcription is rather difficult. One attempt to visualize the emergence of mutations that escaped mismatch repair (MMR) in E. coli, used a plasmid derived functional MutL-GFP fusion in an E. coli strain deficient of its native mutL gene (Elez et al., 2010). MutL is recruited by MutS to the site harboring the misincorporated base and further recruits MutH, an endonuclease cleaving the new DNA. Is the newly synthesized strand already methylated by the Dam methylase, MutH cannot distinguish between the two
Introduction Visualization of emerging mutations
strands anymore and the mismatch cannot be repaired (Schofield and Hsieh, 2003; Kunkel and Erie, 2005). Using MutL-GFP, foci occur when MutL accumulates and MutH does not appear or cannot detect the newly synthesized DNA because of premature methylation resulting in a mutation. The problem of this experiment is the low rate of non-repaired mismatches, in fact only 0.45% of the investigated cells contained a MutL derived GFP focus. Consistent with the previous assumption, a deletion of mutH or the removal of proofreading activity of the DNA polymerase increased the fraction of cells harboring a GFP focus up to 52% (Elez et al., 2010). To conclude, this study investigated the occurrence of mutations randomly distributed over the whole genome and derived from a defective MMR machinery. The low level of foci formation in cells having a functional MMR machinery is not sufficient for practical analyses using for instance double mutants to investigate the influence of
other DNA repair proteins. A system enabling the investigation of a specific mutation in a specific locus on the level of single cells with a functional DNA repair machinery, would substantially contribute to understanding of repair machineries. This can be achieved by the usage of an activator/reporter system (Dormeyer, 2014).
The activator/reporter system provides a mutable unit in form of a transcriptional activator artificially inactivated by a direct repeat within region of the gene important for DNA binding of the resulting protein. The activation of the transcription factor by the precise excision of one repeat unit is comparable to the native situation of the gudBCR gene. The major difference is that the activation of the gudBCR gene is only detectable by the growth advantage conferred by the acquisition of a functional GDH in the absence of RocG. The activation of the
Fig. 1.9 Overview of the activator/reporter system
A: Scheme of the activator/reporter system. The activator unit consists of the constitutively active gudB promoter and the artificially inactivated transcription factor gene prfACR. Upon TR excision PrfA+ activates the plcA derived promoter of the reporter unit harboring the gfp reporter gene and the gudB+ gene conferring a growth advantage on selective medium. B: Scheme of several activator units introduced into the B. subtilis genome increasing the chance of a mutation to occur. C: Activator/reporter strain and emerged SMs on a selective SP plate after 6 dpi RT. Scale 2 mm. D: Cell cultures of the activator/reporter strain (above) and its SM (below) at OD600 1.
Scale 5 µm. E: Western blot analysis of the activator/reporter strain and its SM using α-RocG antibody for the detection of GudB and α-PrfA for the detection of PrfA. (Adapted from Dormeyer, 2014)
gudB+ gfp
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transcription factor results in the expression of the reporter unit consisting of an active gudB+ gene conferring a growth advantage as just described and the gfp reporter gene for visualization. The gudB+ gene allows easy detection of SMs on plate, whereas the gfp gene allows easy detection on the level of single cells.
A similar system exists using an artificial operon, that is under the control of an inactive promoter and expresses upon TR excision the gudB+ gene and the gfp reporter gene (Dormeyer et al., 2014). However, in this system the mutation rate is very low. To enhance the rate of mutations in the activator/reporter system, it is planned to introduce several activator units enabling successful detection of emerging mutations on the level of single cells. The activator/reporter system is only functional in B. subtilis when the activator unit does not interfere with native genes from B. subtilis and when the reporter unit does not exhibit a basal expression that is sufficient to cope with the lack of GDH.
Therefore, a transcription factor promoter pair from L. monocytogenes is used for the activator/reporter system. The major virulence regulator PrfA encoded by prfA, which is fused to the constitutively active promoter of the gudBCR gene, forms the activator unit. The reporter unit is under the control of the promoter from the plcA gene encoding for virulence factor in L. monocytogenes. So far, the activator/reporter system was shown to be functional (Dormeyer, 2014). The activator unit is constitutively expressed and in contrast to the GudBCR protein also detectable in its inactive form via Western blot (Gunka et al., 2012; Dormeyer, 2014).
However, until now the emergence of the TR mutation in the prfACR gene remains to be shown on the level of single cells.
1.6. Objectives
The aim of this thesis is to get a better understanding of how glutamate homeostasis is maintained in B. subtilis. The GDHs from the laboratory B. subtilis strain 168 are of special interest. During growth on rich medium a rocG deficient strain lacking the GDH RocG rapidly forms suppressor mutants that have activated the inactive gudBCR gene by the precise excision of a TR unit (Ch. 1.4). The high frequency of the decryptification suggests the existence of a specific mutational machinery to be involved in the mutagenesis process. Previously, it was shown that the transcription-repair coupling factor Mfd is involved in the mutagenesis of the gudBCR gene. The influence of transcription on TR mutagenesis in general will be investigated using promoters of different strength. Transcription may lead to mutations when the transcription machinery collides with the replication machinery. Therefore, it will be investigated whether the emergence of the mutation is influcenced by the orientation of a gene harboring the TR and by factors participating in the repair of the collision (Ch. 1.4.1.1).
Moreover, GltC mutants lacking the transcriptional activator of the GltAB encoding gltAB genes are auxotrophic for glutamate. It was previously shown that suppressor mutants accumulate, which have acquired the gltR24 mutation enabling the encoded TF GltR24 variant to compensate for the loss of GltC (Belitsky and Sonenshein, 1997). In this thesis, it is planned to assess whether the DNA-binding activity of GltR24 is controlled by the GDHs, as it is the case for GltC. It is also planned to visualize emerging mutations in suppressor mutants at the level of single cells.