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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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Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites. (A) A histogram of the BM amplitude obtained from the 580 bp DNA molecules representing the recombinant products. The average BM value peaks at 43.3 ± 6.8 nm (n = 103). (B) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing parallel loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed but failed to continue through the recombination process. (ii) An example of a molecule that synapsed and continued through the strand-cleavage step to the strand-migration step and then to the strand-ligation steps. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern labels the average BM value of the expected recombinant excision product. (C). Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing parallel loxP sites. The initial substrate is the 1267 bp DNA molecule containing parallel loxP sites and has an average BM amplitude of 80.5 ± 10.5 nm. Two complexes are formed after interaction with Cre recombinase. The first is the synaptic complex that corresponds to the formation of a Cre tetramer with an average BM value of 43.8 ± 8.7 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations, corresponding to the association of Cre dimers with loxP sites with an average BM value of 59.3 ± 6.8 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but failed to complete the recombination process; (ii) a molecule that synapsed and formed either the recombinant product or a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (D) (i) The distribution of the BM amplitude before the addition of Cre recombinase, which shows an average value of 80.5 ± 10.5 nm, is indicated with the letter (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase [59.3 ± 6.8 nm and 43.8 ± 8.7 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively]. (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time (n = 76). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (E) to a value representing the abortive complex state and the association rate constant of (6.8 ± 0.4) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.96, n = 16) were obtained. (F) To a value representing the synapse state, and an association rate constant of (6.1 ± 0.6) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.99 n = 70) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL. (G) BM time-trace of a 1267 bp DNA molecule containing parallel loxP sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and the addition of 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. (i) The BM time trace of a molecule that has successfully formed a synaptic complex following a short-lived presynapse state. (ii) The BM time trace of a molecule that failed to synapse and was trapped in the abortive complex state. For those molecules undergoing BM transitions from the original substrate to the synapse state, leading to an unsuccessful recombination, the short-lived presynapse state duration is classified as type a. For molecules that experience BM transitions from the synapse state to the original substrate, the short-lived presynapse state duration is classified as type b. For those molecules showing decreased BM, even after the SDS challenge, indicating the formation of a stable synapse state, the short-lived presynapse state duration is classified as type c. (H) The dwell times in the abortive complexes formed with the DNA molecules containing parallel loxP sites that failed to synapse during observation were pooled and then fitted to a single exponential decay [R2 = 0.98, Ο„1 = (5.2 ± 1.0) × 10βˆ’3 sβˆ’1, n = 26]. (I) (i)–(iii) The dwell times in the short-lived presynapse state were pooled separately to build dwell-time histograms fitted to a single exponential decay algorithm with values of (7.7 ± 0.1) × 10βˆ’1 sβˆ’1 (n = 17, R2 = 0.99), (6.6 ± 0.4) × 10βˆ’1 sβˆ’1 (n = 16, R2 = 0.97) and (4.2 ± 0.7) × 10βˆ’1 sβˆ’1 (n = 40, R2 = 0.99), representing the association rate constant of unstable synaptic complexes, the dissociation rate constant of presynaptic complexes and the association rate constant of correct synaptic complexes, respectively. The N mentioned above is the number of events observed. The error is within a 95% CL.
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gks274-F2: Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites. (A) A histogram of the BM amplitude obtained from the 580 bp DNA molecules representing the recombinant products. The average BM value peaks at 43.3 ± 6.8 nm (n = 103). (B) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing parallel loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed but failed to continue through the recombination process. (ii) An example of a molecule that synapsed and continued through the strand-cleavage step to the strand-migration step and then to the strand-ligation steps. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern labels the average BM value of the expected recombinant excision product. (C). Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing parallel loxP sites. The initial substrate is the 1267 bp DNA molecule containing parallel loxP sites and has an average BM amplitude of 80.5 ± 10.5 nm. Two complexes are formed after interaction with Cre recombinase. The first is the synaptic complex that corresponds to the formation of a Cre tetramer with an average BM value of 43.8 ± 8.7 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations, corresponding to the association of Cre dimers with loxP sites with an average BM value of 59.3 ± 6.8 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but failed to complete the recombination process; (ii) a molecule that synapsed and formed either the recombinant product or a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (D) (i) The distribution of the BM amplitude before the addition of Cre recombinase, which shows an average value of 80.5 ± 10.5 nm, is indicated with the letter (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase [59.3 ± 6.8 nm and 43.8 ± 8.7 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively]. (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time (n = 76). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (E) to a value representing the abortive complex state and the association rate constant of (6.8 ± 0.4) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.96, n = 16) were obtained. (F) To a value representing the synapse state, and an association rate constant of (6.1 ± 0.6) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.99 n = 70) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL. (G) BM time-trace of a 1267 bp DNA molecule containing parallel loxP sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and the addition of 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. (i) The BM time trace of a molecule that has successfully formed a synaptic complex following a short-lived presynapse state. (ii) The BM time trace of a molecule that failed to synapse and was trapped in the abortive complex state. For those molecules undergoing BM transitions from the original substrate to the synapse state, leading to an unsuccessful recombination, the short-lived presynapse state duration is classified as type a. For molecules that experience BM transitions from the synapse state to the original substrate, the short-lived presynapse state duration is classified as type b. For those molecules showing decreased BM, even after the SDS challenge, indicating the formation of a stable synapse state, the short-lived presynapse state duration is classified as type c. (H) The dwell times in the abortive complexes formed with the DNA molecules containing parallel loxP sites that failed to synapse during observation were pooled and then fitted to a single exponential decay [R2 = 0.98, Ο„1 = (5.2 ± 1.0) × 10βˆ’3 sβˆ’1, n = 26]. (I) (i)–(iii) The dwell times in the short-lived presynapse state were pooled separately to build dwell-time histograms fitted to a single exponential decay algorithm with values of (7.7 ± 0.1) × 10βˆ’1 sβˆ’1 (n = 17, R2 = 0.99), (6.6 ± 0.4) × 10βˆ’1 sβˆ’1 (n = 16, R2 = 0.97) and (4.2 ± 0.7) × 10βˆ’1 sβˆ’1 (n = 40, R2 = 0.99), representing the association rate constant of unstable synaptic complexes, the dissociation rate constant of presynaptic complexes and the association rate constant of correct synaptic complexes, respectively. The N mentioned above is the number of events observed. The error is within a 95% CL.

Mentions: Next, the 1267 bp DNA molecules containing two parallel loxP sites separated by 653 bp were investigated to explore excision recombination. Previous studies have suggested that two Cre recombinases bind to one of the loxP sites, which consists of two recombinase-binding elements (RBEs), and that this process occurs with a high level of cooperation to form the presynaptic complex (13,22). One such Cre-loxP presynaptic complex then interacts with another Cre-loxP presynaptic complex to form a recombinant synaptic complex (13,26). The BM value of the excision product obtained using the 580 bp DNA molecule was determined (43.3 ± 6.8 nm, n = 103, Figure 2A). Examples of single-molecule time traces are shown in Figure 2B(i)–(iii). To confirm whether recombination proceeded or not, the DNA molecules were challenged with 0.05% SDS to disrupt non-covalent Cre–loxP interactions. The change in the BM amplitude of the DNA molecules containing parallel loxP sites can be categorized as either a synapse state or an abortive complex state which is deficient at the Cre–Cre interaction interface in response to the Cre recombinase. After the addition of SDS, there are two possible outcomes for molecules that have entered the synapse state. These outcomes are, first, a return to the original DNA substrate for the molecules that failed to proceed with the catalytic recombination process, as depicted in Figure 2C(i), and second, remaining at a short DNA length status, which corresponds to either the excision recombinant product or a stable Holliday junction intermediate, as depicted in Figure 2C(ii). In contrast, those molecules trapped at abortive complexes eventually returned to the original DNA substrate before the SDS challenge, as depicted in Figure 2C(iii). Among the 143 molecules analyzed with a starting BM amplitude between 70 and 91 nm based on a 40-frame averaging window, only 76 molecules showed a significant change in the BM time trace in response to the addition of Cre recombinase. Among these 76 molecules, 15 molecules showed a decrease in the BM amplitude to the average value of 59.3 ± 6.8 nm within the 95% CL and failed to synapse within the duration of the experiment [Figure 2B(iii) and 2D(ii)]. This magnitude of decrease in the BM amplitude is different from that observed for the excision recombinant product obtained using 580 bp DNA molecules. This scenario indicates the formation of abortive complexes in which two loxP sites are occupied, but with a defect at the Cre–Cre interaction interface. The remainder of the molecules showed a decrease in the BM amplitude to an average value of 43.8 ± 8.7 nm within the 95% CL, similar to that of the expected excision recombinant product, a 580 bp DNA molecule. This observation indicates the formation of synaptic complexes or the excision recombinant product [Figure 2D(ii)]. After 30 min of reaction time, the BM of the DNA molecules that failed to proceed in the catalytic recombination process returned to the starting BM value after the SDS challenge [Figure 2D(iv)]. However, the BM value of some DNA molecules (71.6%) remained low after the SDS challenge, indicating the formation of either excised products or a stable Holliday junction intermediate [Figure 2D(iv)]. The dwell times between the addition of Cre and the shortening of the DNA molecules containing parallel loxP sites were classified into two groups: one for molecules that will be trapped in an abortive complex and the other for molecules that will form the synaptic complex quickly with a short-lived presynaptic complex. Then, the dwell time histograms were fitted to a single exponential model (Figure 2E and 2F) with association rate constants of (6.8 ± 0.4) × 104 Mβˆ’1 sβˆ’1 and (6.1 ± 0.6) × 104 Mβˆ’1 sβˆ’1 for the formation of an abortive complex and the synapse state, respectively. A total of 15 of 76 DNA molecules containing parallel loxP sites were trapped in the abortive complex [Figure 2G(i)]. The distribution of the durations of abortive complexes was fitted to a single exponential model, and an abortive complex dissociation rate constant of (5.2 ± 1.0) × 10βˆ’3 sβˆ’1 was obtained (Figure 2H). For those molecules exhibiting BM transitions from the original substrate to the synapse state [Figure 2G(ii)], leading to unsuccessful recombination, the duration of the short-lived presynapse state is classified as type a, representing the formation of unstable synaptic complexes. For molecules exhibiting BM transitions from the synapse state to the original substrate, the duration of the short-lived presynapse state is classified as type b, representing the dissociation of short-lived presynaptic complexes. For molecules presenting decreased BM, even after an SDS challenge, indicating the formation of stable synaptic complexes, the duration of the short-lived presynapse state is classified as type c, which represents the formation of stable synaptic complexes. As a result, all the durations of the short-lived presynapse states were categorized and used to construct separate histograms with a bin size of 1 s [Figure 2I(i–iii)]. The histograms were fitted to a single exponential model with rate constants of (7.7 ± 0.1) × 10βˆ’1 sβˆ’1, (6.6 ± 0.4) × 10βˆ’1 sβˆ’1 and (4.2 ± 0.7) × 10βˆ’1 sβˆ’1, representing the formation of unstable synaptic complexes, the decomposition of unstable presynaptic complexes and the formation of stable synaptic complexes of DNA molecules that successfully synapse during the experimental period, respectively.Figure 2.


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)

Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites. (A) A histogram of the BM amplitude obtained from the 580 bp DNA molecules representing the recombinant products. The average BM value peaks at 43.3 ± 6.8 nm (n = 103). (B) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing parallel loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed but failed to continue through the recombination process. (ii) An example of a molecule that synapsed and continued through the strand-cleavage step to the strand-migration step and then to the strand-ligation steps. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern labels the average BM value of the expected recombinant excision product. (C). Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing parallel loxP sites. The initial substrate is the 1267 bp DNA molecule containing parallel loxP sites and has an average BM amplitude of 80.5 ± 10.5 nm. Two complexes are formed after interaction with Cre recombinase. The first is the synaptic complex that corresponds to the formation of a Cre tetramer with an average BM value of 43.8 ± 8.7 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations, corresponding to the association of Cre dimers with loxP sites with an average BM value of 59.3 ± 6.8 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but failed to complete the recombination process; (ii) a molecule that synapsed and formed either the recombinant product or a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (D) (i) The distribution of the BM amplitude before the addition of Cre recombinase, which shows an average value of 80.5 ± 10.5 nm, is indicated with the letter (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase [59.3 ± 6.8 nm and 43.8 ± 8.7 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively]. (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time (n = 76). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (E) to a value representing the abortive complex state and the association rate constant of (6.8 ± 0.4) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.96, n = 16) were obtained. (F) To a value representing the synapse state, and an association rate constant of (6.1 ± 0.6) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.99 n = 70) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL. (G) BM time-trace of a 1267 bp DNA molecule containing parallel loxP sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and the addition of 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. (i) The BM time trace of a molecule that has successfully formed a synaptic complex following a short-lived presynapse state. (ii) The BM time trace of a molecule that failed to synapse and was trapped in the abortive complex state. For those molecules undergoing BM transitions from the original substrate to the synapse state, leading to an unsuccessful recombination, the short-lived presynapse state duration is classified as type a. For molecules that experience BM transitions from the synapse state to the original substrate, the short-lived presynapse state duration is classified as type b. For those molecules showing decreased BM, even after the SDS challenge, indicating the formation of a stable synapse state, the short-lived presynapse state duration is classified as type c. (H) The dwell times in the abortive complexes formed with the DNA molecules containing parallel loxP sites that failed to synapse during observation were pooled and then fitted to a single exponential decay [R2 = 0.98, Ο„1 = (5.2 ± 1.0) × 10βˆ’3 sβˆ’1, n = 26]. (I) (i)–(iii) The dwell times in the short-lived presynapse state were pooled separately to build dwell-time histograms fitted to a single exponential decay algorithm with values of (7.7 ± 0.1) × 10βˆ’1 sβˆ’1 (n = 17, R2 = 0.99), (6.6 ± 0.4) × 10βˆ’1 sβˆ’1 (n = 16, R2 = 0.97) and (4.2 ± 0.7) × 10βˆ’1 sβˆ’1 (n = 40, R2 = 0.99), representing the association rate constant of unstable synaptic complexes, the dissociation rate constant of presynaptic complexes and the association rate constant of correct synaptic complexes, respectively. The N mentioned above is the number of events observed. The error is within a 95% CL.
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gks274-F2: Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites. (A) A histogram of the BM amplitude obtained from the 580 bp DNA molecules representing the recombinant products. The average BM value peaks at 43.3 ± 6.8 nm (n = 103). (B) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing parallel loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed but failed to continue through the recombination process. (ii) An example of a molecule that synapsed and continued through the strand-cleavage step to the strand-migration step and then to the strand-ligation steps. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern labels the average BM value of the expected recombinant excision product. (C). Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing parallel loxP sites. The initial substrate is the 1267 bp DNA molecule containing parallel loxP sites and has an average BM amplitude of 80.5 ± 10.5 nm. Two complexes are formed after interaction with Cre recombinase. The first is the synaptic complex that corresponds to the formation of a Cre tetramer with an average BM value of 43.8 ± 8.7 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations, corresponding to the association of Cre dimers with loxP sites with an average BM value of 59.3 ± 6.8 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but failed to complete the recombination process; (ii) a molecule that synapsed and formed either the recombinant product or a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (D) (i) The distribution of the BM amplitude before the addition of Cre recombinase, which shows an average value of 80.5 ± 10.5 nm, is indicated with the letter (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase [59.3 ± 6.8 nm and 43.8 ± 8.7 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively]. (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time (n = 76). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (E) to a value representing the abortive complex state and the association rate constant of (6.8 ± 0.4) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.96, n = 16) were obtained. (F) To a value representing the synapse state, and an association rate constant of (6.1 ± 0.6) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.99 n = 70) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL. (G) BM time-trace of a 1267 bp DNA molecule containing parallel loxP sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and the addition of 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. (i) The BM time trace of a molecule that has successfully formed a synaptic complex following a short-lived presynapse state. (ii) The BM time trace of a molecule that failed to synapse and was trapped in the abortive complex state. For those molecules undergoing BM transitions from the original substrate to the synapse state, leading to an unsuccessful recombination, the short-lived presynapse state duration is classified as type a. For molecules that experience BM transitions from the synapse state to the original substrate, the short-lived presynapse state duration is classified as type b. For those molecules showing decreased BM, even after the SDS challenge, indicating the formation of a stable synapse state, the short-lived presynapse state duration is classified as type c. (H) The dwell times in the abortive complexes formed with the DNA molecules containing parallel loxP sites that failed to synapse during observation were pooled and then fitted to a single exponential decay [R2 = 0.98, Ο„1 = (5.2 ± 1.0) × 10βˆ’3 sβˆ’1, n = 26]. (I) (i)–(iii) The dwell times in the short-lived presynapse state were pooled separately to build dwell-time histograms fitted to a single exponential decay algorithm with values of (7.7 ± 0.1) × 10βˆ’1 sβˆ’1 (n = 17, R2 = 0.99), (6.6 ± 0.4) × 10βˆ’1 sβˆ’1 (n = 16, R2 = 0.97) and (4.2 ± 0.7) × 10βˆ’1 sβˆ’1 (n = 40, R2 = 0.99), representing the association rate constant of unstable synaptic complexes, the dissociation rate constant of presynaptic complexes and the association rate constant of correct synaptic complexes, respectively. The N mentioned above is the number of events observed. The error is within a 95% CL.
Mentions: Next, the 1267 bp DNA molecules containing two parallel loxP sites separated by 653 bp were investigated to explore excision recombination. Previous studies have suggested that two Cre recombinases bind to one of the loxP sites, which consists of two recombinase-binding elements (RBEs), and that this process occurs with a high level of cooperation to form the presynaptic complex (13,22). One such Cre-loxP presynaptic complex then interacts with another Cre-loxP presynaptic complex to form a recombinant synaptic complex (13,26). The BM value of the excision product obtained using the 580 bp DNA molecule was determined (43.3 ± 6.8 nm, n = 103, Figure 2A). Examples of single-molecule time traces are shown in Figure 2B(i)–(iii). To confirm whether recombination proceeded or not, the DNA molecules were challenged with 0.05% SDS to disrupt non-covalent Cre–loxP interactions. The change in the BM amplitude of the DNA molecules containing parallel loxP sites can be categorized as either a synapse state or an abortive complex state which is deficient at the Cre–Cre interaction interface in response to the Cre recombinase. After the addition of SDS, there are two possible outcomes for molecules that have entered the synapse state. These outcomes are, first, a return to the original DNA substrate for the molecules that failed to proceed with the catalytic recombination process, as depicted in Figure 2C(i), and second, remaining at a short DNA length status, which corresponds to either the excision recombinant product or a stable Holliday junction intermediate, as depicted in Figure 2C(ii). In contrast, those molecules trapped at abortive complexes eventually returned to the original DNA substrate before the SDS challenge, as depicted in Figure 2C(iii). Among the 143 molecules analyzed with a starting BM amplitude between 70 and 91 nm based on a 40-frame averaging window, only 76 molecules showed a significant change in the BM time trace in response to the addition of Cre recombinase. Among these 76 molecules, 15 molecules showed a decrease in the BM amplitude to the average value of 59.3 ± 6.8 nm within the 95% CL and failed to synapse within the duration of the experiment [Figure 2B(iii) and 2D(ii)]. This magnitude of decrease in the BM amplitude is different from that observed for the excision recombinant product obtained using 580 bp DNA molecules. This scenario indicates the formation of abortive complexes in which two loxP sites are occupied, but with a defect at the Cre–Cre interaction interface. The remainder of the molecules showed a decrease in the BM amplitude to an average value of 43.8 ± 8.7 nm within the 95% CL, similar to that of the expected excision recombinant product, a 580 bp DNA molecule. This observation indicates the formation of synaptic complexes or the excision recombinant product [Figure 2D(ii)]. After 30 min of reaction time, the BM of the DNA molecules that failed to proceed in the catalytic recombination process returned to the starting BM value after the SDS challenge [Figure 2D(iv)]. However, the BM value of some DNA molecules (71.6%) remained low after the SDS challenge, indicating the formation of either excised products or a stable Holliday junction intermediate [Figure 2D(iv)]. The dwell times between the addition of Cre and the shortening of the DNA molecules containing parallel loxP sites were classified into two groups: one for molecules that will be trapped in an abortive complex and the other for molecules that will form the synaptic complex quickly with a short-lived presynaptic complex. Then, the dwell time histograms were fitted to a single exponential model (Figure 2E and 2F) with association rate constants of (6.8 ± 0.4) × 104 Mβˆ’1 sβˆ’1 and (6.1 ± 0.6) × 104 Mβˆ’1 sβˆ’1 for the formation of an abortive complex and the synapse state, respectively. A total of 15 of 76 DNA molecules containing parallel loxP sites were trapped in the abortive complex [Figure 2G(i)]. The distribution of the durations of abortive complexes was fitted to a single exponential model, and an abortive complex dissociation rate constant of (5.2 ± 1.0) × 10βˆ’3 sβˆ’1 was obtained (Figure 2H). For those molecules exhibiting BM transitions from the original substrate to the synapse state [Figure 2G(ii)], leading to unsuccessful recombination, the duration of the short-lived presynapse state is classified as type a, representing the formation of unstable synaptic complexes. For molecules exhibiting BM transitions from the synapse state to the original substrate, the duration of the short-lived presynapse state is classified as type b, representing the dissociation of short-lived presynaptic complexes. For molecules presenting decreased BM, even after an SDS challenge, indicating the formation of stable synaptic complexes, the duration of the short-lived presynapse state is classified as type c, which represents the formation of stable synaptic complexes. As a result, all the durations of the short-lived presynapse states were categorized and used to construct separate histograms with a bin size of 1 s [Figure 2I(i–iii)]. The histograms were fitted to a single exponential model with rate constants of (7.7 ± 0.1) × 10βˆ’1 sβˆ’1, (6.6 ± 0.4) × 10βˆ’1 sβˆ’1 and (4.2 ± 0.7) × 10βˆ’1 sβˆ’1, representing the formation of unstable synaptic complexes, the decomposition of unstable presynaptic complexes and the formation of stable synaptic complexes of DNA molecules that successfully synapse during the experimental period, respectively.Figure 2.

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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