Archive for March, 2012

The Amazing History of DR397

Thursday, March 29th, 2012

Drew Reams studies unselected duplication formation, and recently found an eel in the DNA!

Recall that duplication formation is a fundamental small-effect driver of evolutionary adaptation, and TIDs may be the primum mobile of most or all duplications. Drew’s “recalcitrant” duplication strains — those with joints not easily determined by multiplex PCR — are a class which should include TIDs. To analyze them, he sequences their genomes by Illumina technology.

We think TID formation often begins with a snap-back of a 3′ end at a short inverted repeat, forming a stem-loop structure which then initiates DNA synthesis using self as template.

A subsequent template switch restores the fork and finishes the TID. See the excellent article in our Encyclopedia.

The extensive secondary structures at such symmetric TID joints are toxic and rarely observed. Instead, remodelling asymmetric deletions are selected spontaneously, yielding “SJ” (“short junction”) joints. David Leach has shown that sbcCD backgrounds tolerate such structures by not cutting them with the endonuclease product.

Drew wondered what would happen to duplication frequencies in such backgrounds, which may allow the cell to survive the two initial steps of TID formation and increase the yield of duplications. To enhance stem-loop persistance, he also made the cells recQ to prevent stem melting.

He observed a 2-fold increase in duplications in both the chromosome and wild type F’128. The latter contains two IS3 elements and nearly all spontaneous duplications happen by recombination between them. If at least one were removed, the duplication frequency increased by an order of magnitude compared to sbcCD+ recQ+ cells. Something definitely happens in the absence of sbcCD and recQ when IS recombination is blocked

One sequenced duplication was  in F’128 of strain DR397. It had a lacZ read depth 3-fold greater than the chromosome with remaining plasmid DNA about 9-fold greater. In addition, there was a large deletion extending from traI up to lacZ accounting for ~20% of the plasmid.

When we inspected the anomalous read-pair data, we discovered two symmetrical TID joins. We were able to confirm these by showing that reads right at the edge of the deletion window fully contained these joints.

Drew’s suspicions were vindicated. Removing activities which cut stem loop structures or prevent them from folding encourages secondary structure formation and reduces counterselection of the long symmetrical products of snap back and strand switching.

But, what’s the meaning of the enormous deletion? There are a couple of models which come to mind.

Model 1: A snapback at traI followed by another at lac, 5′ resection and ligation of ends will produce a product that matches our observations. It would look something like the following: 

Model 2: A snapback at traI and strand switch at lac will restore the replication fork and lead to a TID. Eventual remodelling by recombination of the flanking repeats will yield our observed product: 

Other related models are possible, with the order of the snapbacks and strand switches reversed, with double strand switches instead of snapbacks, etc. Nevertheless, we favor the second model above, as it only involves two steps to get the essential intermediate, while many generations can pass before selection of the final product.

An interesting aspect of the TID model is the inevitability of the remodeling event. When the origin is itself in the TID, counterselection on a large number of unnecessary genes leads easily to their deletion by recombination. The resulting fitness increase will lead to expansion in the population of the symmetric inversion and eventual extinction of the TID.

-- Eric Kofoid

Sophie’s Mystery

Wednesday, March 28th, 2012

We’ve been developing methods to identify cells carrying TIDs (“Tandem Inverted Duplications”). Sophie Maisnier-Patin uses P22 transduction to introduce various tools which will introduce a drug resistance cassette only if they are able to recombine into such inverted structures.

Sophie asks whether a kanR MudJ element can nucleate TIDs by virtue of its terminal inverted repeat? This entity is 104 bp long and can form a perfectly paired 48 bp stem with an 8 base loop. The hypothesis is something like the following:

The tool Sophie used in this experiment contained two oppositely facing MudJ elements:

She transduced this into a recipient containing MudJ and a deletion which should prevent recombination with cob and his. :

The resulting strain, SMP1666, should have a lac+ camR kanR trp- phenotype. Unfortunately, no TIDs was found. Instead, Illumina and subsequent PCR analyses showed that one end of the tool inserted by a strand annealing mechanism, as the his:cob deletion was not quite large enough. The strain was unstable for chloramphenicol resistance, expected from the flanking trpB::MudJ direct repeats. Sophie showed by transduction to trp+ that the SMP1666 parent, as expected, contained a MudJ-bisected trpB locus, and that trp+transductants, whether camR or camS, were invariably lac-, another prediction of the hypothesis. The corrected model became:

The Mystery

A large fraction of the trp+ lac- transductants retained kanamycin resistance! How could this be, when elimination of the last MudJ in the chromosome should, by definition, also remove the remaining kanR and lac loci? Clearly, something was wrong. There had to be another MudJ with an impaired promoter incapable of driving lacZ. We wrestled for weeks with elaborate models. All of them suffered from the need for at least two concerted events, implying a low rearrangement frequency, when the opposite had been observed.

We remembered some interesting results from two other Sophian strains, SMP1750 & TT26263, in which the donor DNA had recombined with the recipient through the MudJ inverted repeat locus itself, even though the element orientations were divergent. Although the inverted-repeat region is short, when folded it is prone to attack by SbcCD. This would provide many 3′ ends which could then anneal and rescue the cell. A corollary is that the MudJ stem-loop structure is recombinationally potent.

In light of this, we have refined our model:

The head-to-head Muds in the middle would have no promoter at all for lac, but the internal constitutive kanR promoter would still be active. The trpB gene would, of course, have been restored exactly as expected in earlier models.

-- Eric Kofoid