IS200/IS605 Family-Associated TnpB Increases Transposon Activity and Retention

Reviewer #1 (Public Review):

Summary:
The nuclease protein TnpB is ubiquitous across microorganisms. It is associated with the stabilization of transposable genetic elements and is a putative precursor of the RNA-guided endonuclease Cas9 from the CRISPR system. Despite its potential as a gene-editing tool, TnpB is not well understood. In this study, the authors use a fluorescence-based construct to individually quantify the transposable-element excision rate and the abundance of the two ancillary proteins TnpA and TnpB. They develop a mathematical model describing the role of TnpB in transposable-element stabilization.

Strengths:
The methodology (with schematic shown in Figure 1A) is powerful and sophisticated. The authors are able to de-convolve excision events from the individual abundances of TnpA and TnpB.

Weaknesses:
The claim that TnpB expression level correlates positively (and significantly) with the probability of a growth-disrupting integration (called 'b') is not well-supported by the data shown in Fig. 3D. The modelling of results shown in Figure 4 are not tied directly to the experimental data shown in Figures 1-3.

Reviewer #2 (Public Review):

Summary:
Dr. Khulman and colleagues present a very interesting experimental and mathematical modeling work on IS608 transposition. The system has a number of unique advantages that create experimental possibilities utilized here to investigate transposition dynamics. This kind of approach is badly missing from the field and I believe the experiments and modeling work shown here and the type of results that can be derived are groundbreaking and certainly deserve high visibility.

Strengths:
The attempt to measure and model transposition dynamics in cells.

Weaknesses:
- Lack of controls using an active site mutant of TnpB.
- Lack of control in RecA- cells as transposon restoration is proposed to be dependent on homologous recombination.
- Lack of consideration of the levels of ωRNA present.

Reviewer #3 (Public Review):

The authors use a set of reporter assays to probe the impact of TnpB on IS605 transposition. This is an innovative approach to studying transposition that will be of interest to other groups. The authors conclude that TnpB likely has two activities: (ii) promoting "homing", where the transposon is restored following excision, and (ii) promoting the transposition activity of the transposase TnpA. The paper provides an independent validation of the recently reported homing activity of TnpB, where the transposon is restored following excision, and suggests an additional function for TnpB in enhancing the transposase activity of the TnpA transposase.

Strengths
- The innovative use of reporter assays is an excellent way to infer the details of transposition, making a convincing case that TnpB plays two distinct roles.

Weaknesses
- The authors need to discuss their conclusions in light of a very relevant recent paper from the Sternberg group demonstrating a role for TnpB in homing.
- There doesn't appear to be an assessment of how well the model fits the experimental data, or any attempt to test how accurately the model can predict the effects of perturbing the system.
- The figures could be presented more clearly.