Trying to reconcile the PC-PG phenotype

I recall one of the first experiments that Paul Hurd carried out on mutants at the Pro-Cys element (motif IV) in order to get to the bottom of the pyrimidinone data that we had been obtaining, thanks to some lovely chemical synthesis by Bernard Connolly. Briefly, it was difficult to explain whether the oligos containing pyrimidinone in place of the target C in CCGG sites, was eliciting covalent adduct formation with M.MspI. We wanted to demonstrate that this was Cys dependent (with an outside possibility that the pyrimidinone could be sufficiently reactive to surrender to an attack by Ser). Anyway, for completeness, we decided to make the PC-PG mutant as well as several others, since Rich Roberts had shown anomalies with the Gly substitution, and Ashok Bhagwat had seen similar behaviour with M.EcoRII. Probably the most surprising result turned out to be the viability of the M.MspI C81G mutant (using M.HhaI numbering). Or that's what we thought.....

What we hadn't appreciated was the impact of the cloning vector, or rather the consequences of fusing our M.MspI ORF in frame with GST (we were routinely using pGEX vectors), since fusions to GST seemed to stabilise the expressed methylase enzymes, made them easy to purify the GST seemed to be tolerated well, so we generally didn't bother removing it in gel shift assays. However, GST is a homodimer (left). When Paul sequenced the apparently viable clones, we realised that something was amiss. In fact, we took a while to realise what had happened, since SDS PAGE analysis of the expressed ProGly mutant ran in its expected molecular weight position. The sequence analysis revealed that the first set of clones had an in frame 30bp deletion which took out ten amino acids at the active site, including the Pro-Gly sequence! Over the next few years, we investigated different methylase genes and used different host strains. In all cases, the induction of plasmid mutations turned out to be dependent on the presence of a dimerisation domain and led to insertions, deletions or mobilisation of transposable elements etc. All mutants eliminated the function of the ProGly mutant.

Having tried to rationalise the observation for a number of years and looked into copy number issues etc., it occurred to me that these observations have some resonance with chromothripsis. This relatively recent phenomenon, in which it has been suggested that a catastrophic replication associated event leads to massive genetic mutation (insertions, deletions etc), could provide some insight. If we sequence the genomes of several Pro-Gly transformants and compare them with standard plasmid transformants, we should see whether the error prone repair is plasmid directed or a generalized error prone mechanism/s. The results will enable us to plan our next set of experiments more strategically, if nothing else! The questions I ask, is, have we developed a bacterial model for chromothripsis? Could it be applicable as a general mutagenesis strategy?