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Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination.

Fan HF - Nucleic Acids Res. (2012)

Bottom Line: Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed.Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined.Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, 112, Taiwan. hffan2@ym.edu.tw

ABSTRACT
Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.

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Related in: MedlinePlus

Mechanism by which bacteriophage P1 Cre recombinase mediates site-specific recombination. The rate constant for each step was obtained via either single exponential decay fitting or bi-exponential decay fitting to the duration distribution histograms. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent the Cre recombinases. The small black dots represent the covalent phosphotyrosine linkage that is created after DNA cleavage. All the processes within the frame—from strand cleavage to strand migration to strand ligation—were assigned as catalytic recombination processes.
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gks274-F7: Mechanism by which bacteriophage P1 Cre recombinase mediates site-specific recombination. The rate constant for each step was obtained via either single exponential decay fitting or bi-exponential decay fitting to the duration distribution histograms. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent the Cre recombinases. The small black dots represent the covalent phosphotyrosine linkage that is created after DNA cleavage. All the processes within the frame—from strand cleavage to strand migration to strand ligation—were assigned as catalytic recombination processes.

Mentions: Using the above kinetic results, the kinetic details of the reaction pathway from beginning to end can be delineated (Figure 7). In a previous single-molecule study on the tyrosine family recombinase λ Int, it was found that presynaptic complexes formed at a rate constant >0.05 s−1, and no detectable dissociation process can be observed within 60 min of reaction time (19). Moreover, it has been found that the interaction between Cre and loxP occurs very rapidly, with a rate constant of ∼108 M−1 s−1 (31). However, the association rate constant of (6.8 ± 0.4) × 104 M−1 s−1 obtained via TPM is slower compared to previous results. It is believed that Cre recombinase is monomeric in solution and binds to the loxP site one by one to form a dimer (21–23). However, only the presynapse state in which two loxP sites are occupied by Cre recombinases can be differentiated because of the limited resolution of TPM. According to our data, the kAssociation value observed is the rate constant of abortive complex formation, rather than that of DNA binding. Therefore, it cannot be ruled out that the measured association rate constant is underestimated. For these abortive complexes, the dissociation rate constant of Cre recombinase from the loxP site is (5.2 ± 1.0) × 10−3 s−1, which is consistent with previous results (31). The reason for the formation of abortive complexes could be either that there are too few Cre recombinases at the loxP site or that there are one or more inactive Cre recombinases occupying the loxP site. However, an alternative interpretation that cannot be ruled out is that this 19.7% of the population of abortive complexes, results from inactive Cre molecules formed during preparation of the Cre enzyme. In spite of the observed formation of abortive complexes, 61 of 76 molecules formed synaptic complexes rapidly from short-lived presynaptic complexes, and rate constants of (7.7 ± 0.1) × 10−1 s−1 and (4.2 ± 0.7) × 10−1 s−1 were obtained for the formation of wayward complexes and stable synaptic complexes, respectively. It has been reported that this process, which requires a collision between DNA partners, takes place relatively slowly (31,46). However, rapid synaptic behavior has been observed in both the λ Int and Cre recombinase systems (12,19,31). The similarity between these two tyrosine family recombinases reinforces the hypothesis that there are no intrinsic mechanistic properties that cause synapsis to be a step that occurs slowly in vitro. However, the discrepancy between the TPM results and in vivo observations could be explained by the in vivo synapsis process being either intermolecular or involving a much larger separation between the recombining sites, making synapsis a slower process in vivo.


Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination.

Fan HF - Nucleic Acids Res. (2012)

Mechanism by which bacteriophage P1 Cre recombinase mediates site-specific recombination. The rate constant for each step was obtained via either single exponential decay fitting or bi-exponential decay fitting to the duration distribution histograms. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent the Cre recombinases. The small black dots represent the covalent phosphotyrosine linkage that is created after DNA cleavage. All the processes within the frame—from strand cleavage to strand migration to strand ligation—were assigned as catalytic recombination processes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401459&req=5

gks274-F7: Mechanism by which bacteriophage P1 Cre recombinase mediates site-specific recombination. The rate constant for each step was obtained via either single exponential decay fitting or bi-exponential decay fitting to the duration distribution histograms. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent the Cre recombinases. The small black dots represent the covalent phosphotyrosine linkage that is created after DNA cleavage. All the processes within the frame—from strand cleavage to strand migration to strand ligation—were assigned as catalytic recombination processes.
Mentions: Using the above kinetic results, the kinetic details of the reaction pathway from beginning to end can be delineated (Figure 7). In a previous single-molecule study on the tyrosine family recombinase λ Int, it was found that presynaptic complexes formed at a rate constant >0.05 s−1, and no detectable dissociation process can be observed within 60 min of reaction time (19). Moreover, it has been found that the interaction between Cre and loxP occurs very rapidly, with a rate constant of ∼108 M−1 s−1 (31). However, the association rate constant of (6.8 ± 0.4) × 104 M−1 s−1 obtained via TPM is slower compared to previous results. It is believed that Cre recombinase is monomeric in solution and binds to the loxP site one by one to form a dimer (21–23). However, only the presynapse state in which two loxP sites are occupied by Cre recombinases can be differentiated because of the limited resolution of TPM. According to our data, the kAssociation value observed is the rate constant of abortive complex formation, rather than that of DNA binding. Therefore, it cannot be ruled out that the measured association rate constant is underestimated. For these abortive complexes, the dissociation rate constant of Cre recombinase from the loxP site is (5.2 ± 1.0) × 10−3 s−1, which is consistent with previous results (31). The reason for the formation of abortive complexes could be either that there are too few Cre recombinases at the loxP site or that there are one or more inactive Cre recombinases occupying the loxP site. However, an alternative interpretation that cannot be ruled out is that this 19.7% of the population of abortive complexes, results from inactive Cre molecules formed during preparation of the Cre enzyme. In spite of the observed formation of abortive complexes, 61 of 76 molecules formed synaptic complexes rapidly from short-lived presynaptic complexes, and rate constants of (7.7 ± 0.1) × 10−1 s−1 and (4.2 ± 0.7) × 10−1 s−1 were obtained for the formation of wayward complexes and stable synaptic complexes, respectively. It has been reported that this process, which requires a collision between DNA partners, takes place relatively slowly (31,46). However, rapid synaptic behavior has been observed in both the λ Int and Cre recombinase systems (12,19,31). The similarity between these two tyrosine family recombinases reinforces the hypothesis that there are no intrinsic mechanistic properties that cause synapsis to be a step that occurs slowly in vitro. However, the discrepancy between the TPM results and in vivo observations could be explained by the in vivo synapsis process being either intermolecular or involving a much larger separation between the recombining sites, making synapsis a slower process in vivo.

Bottom Line: Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed.Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined.Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, 112, Taiwan. hffan2@ym.edu.tw

ABSTRACT
Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.

Show MeSH
Related in: MedlinePlus