MuA Transposase Separates DNA Sequence Recognition from Catalysis

Goldhaber-Gordon, Ilana; Early, Michael H.; Baker, Tania A.

doi:10.1021/bi035360o

Cited by 9 publications

(7 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Internal primers, annealing within CmR , were combined with R1R2 (ref. 45 ) primers that introduced modifications (see Supplementary Fig. 1b for modifications and Supplementary Table 1 for primers).…”

Section: Methodsmentioning

confidence: 99%

Rapid construction of metabolite biosensors using domain-insertion profiling

et al. 2016

View full text Add to dashboard Cite

Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery.

show abstract

Section: Methodsmentioning

confidence: 99%

Rapid construction of metabolite biosensors using domain-insertion profiling

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Other DNA-binding proteins have modular designs that physically separate the DNA-binding and catalytic roles to some degree. For example, Mu transposase discriminates among most substrates at the binding rather than the catalytic step, presumably because sequence-specific DNA binding and catalysis are carried out by different domains (20). In Flp, it is not clear if sequence discrimination takes place during the initial binding step, in the cleavage step, or a combination of the two.…”

mentioning

confidence: 99%

Binding and Catalytic Contributions to Site Recognition by Flp Recombinase

Whiteson

Rice

2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Flp catalyzes site-specific recombination in a highly sequence-specific manner despite making few direct contacts to the bases within its binding site. Sequence discrimination could take place in the binding and/or the catalytic steps. In this study, we independently measure the binding affinity and initial cleavage rate of Flp recombinase with ϳ20 designed alternate target DNA sequences. Our results show that Flp specificity is largely, although not entirely, imparted at the binding step and is the result of a combination of direct and indirect readout. The Flp binding site includes an A/T-rich region that displays a characteristically narrow minor groove. We find that many A 3 T changes are tolerated at the binding step, whereas C or G substitutions tend to decrease binding affinity. The effects of the latter can be alleviated by replacing guanine with inosine, which removes the N2 amino group that protrudes into the minor groove. Some A 3 T changes reduce binding affinity, due to clashing with nearby residues, reinforcing that specificity requires avoiding negative contacts as well as creating positive ones. A tracts, which can lead to unusually rigid DNA structure, are tolerated during the binding step when placed within the region where the minor groove is already narrow. However, most A tracts slow catalysis more than C or G substitutions. Understanding what kind of sequence variation is tolerated in the binding and catalytic steps helps us understand how the target DNA is recognized by Flp and will be useful in guiding the design of Flp variants with altered specificities.Specificity in protein-DNA interactions is required for many cellular processes including transcription, DNA repair, and recombination. Proteins that modify DNA must distinguish their substrates from an overwhelming number of competing DNA sequences. In many cases, DNA-binding proteins recognize a specific DNA sequence even when there are few direct, unique contacts between the DNA bases and the amino acid side chains. Whereas it was once thought that unique hydrogen bond donor and acceptor patterns in the major groove would be the most important feature in DNA-protein recognition, further studies have revealed that other sequence-specific features of DNA often play a critical role. A large number of DNAbinding proteins have been shown to use indirect readout in their recognition of the appropriate DNA sequence, including the 434 repressor, TATA-binding protein, restriction enzymes including EcoRI, and the DNA bending proteins IHF and HU (1-6).In this study we focus on understanding substrate recognition in a site-specific DNA recombinase. Flp is a tyrosine recombinase from Saccharomyces cerevisiae that specifically recognizes a pair of 13-bp inverted repeats and catalyzes recombination of the intervening sequence. The recombination reaction involves two rounds of DNA cleavage, strand exchange and religation (7). Flp is widely used as a biotechnology tool to knock out genes, especially in Drosophila. Efforts to design recombinases with a...

show abstract

“…Because MuB is a strong stimulator of strand transfer complex formation, 23,35,36 we considered that it might have a specific role in preventing disintegration. To address this question, we devel- Denaturing gel of transposition products after the second incubation.…”

Section: The Mub Activator Promotes Joining and Antagonizes Reversalmentioning

confidence: 99%

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

Lemberg

Schweidenback

Baker

2007

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

DNA transposases use a single active center to sequentially cleave the transposable element DNA and join this DNA to a target site. Recombination requires controlled conformational changes within the transposase to ensure that these chemically distinct steps occur at the right time and place, and that the reaction proceeds in the net forward direction. Mu transposition is catalyzed by a stable complex of MuA transposase bound to paired Mu DNA ends (a transpososome). We find that Mu transpososomes efficiently catalyze disintegration when recombination on one end of the Mu DNA is blocked. The MuB activator protein controls the integration versus disintegration equilibrium. When MuB is present, disintegration occurs slowly and transpososomes that have disintegrated catalyze subsequent rounds of recombination. In the absence of MuB, disintegration goes to completion. These results together with experiments mapping the MuA-MuB contacts during DNA joining suggest that MuB controls progression of recombination by specifically stabilizing a concerted transition to the "joining" configuration of MuA. Thus, we propose that MuB's interaction with the transpososome actively promotes coupled joining of both ends of the element DNA into the same target site and may provide a mechanism to antagonize formation of single-end transposition products.

show abstract

MuA Transposase Separates DNA Sequence Recognition from Catalysis

Cited by 9 publications

References 70 publications

Rapid construction of metabolite biosensors using domain-insertion profiling

Rapid construction of metabolite biosensors using domain-insertion profiling

Binding and Catalytic Contributions to Site Recognition by Flp Recombinase

The Dynamic Mu Transpososome: MuB Activation Prevents Disintegration

Contact Info

Product

Resources

About