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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

Complex formation and recombination events for 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreK201A. (A) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel loxP sites in response to the addition of CreK201A. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. (B) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 78.8 ± 10.2 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (61.0 ± 1.3 nm and 44.0 ± 12.0 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude at 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge at 30 min of incubation time (n = 43). (C) The distribution of the dwell times between recombinase addition and the change in the BM amplitude to (i) a value that represented the abortive complex state and an association rate constant of (2.0 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 16) and to (ii) a value that represented the synapse state and an association rate constant of (1.5 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 121) were obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules observed. (D) (i) The dwell times in the abortive complex state for the DNA molecules containing parallel loxP sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [R2 = 0.98, τ1 = (4.0 ± 0.7) × 10−3 s−1, n = 7]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [R2 = 0.98, k1 = (6.8 ± 0.8) × 10−3 s−1, n = 148] or a bi-exponential decay algorithm [R2 = 1.00, k1 = (4.1 ± 1.9) × 10−2 s−1, A1 = 0.90; k2 = (2.6 ± 0.2) × 10−3 s−1, A2 = 0.10, n = 148]. Solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL. (E) The mechanism through which bacteriophage P1 CreK201A mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent Cre recombinases. The arrows on the DNA molecule indicate the parallel loxP sites.
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gks274-F6: Complex formation and recombination events for 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreK201A. (A) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel loxP sites in response to the addition of CreK201A. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. (B) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 78.8 ± 10.2 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (61.0 ± 1.3 nm and 44.0 ± 12.0 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude at 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge at 30 min of incubation time (n = 43). (C) The distribution of the dwell times between recombinase addition and the change in the BM amplitude to (i) a value that represented the abortive complex state and an association rate constant of (2.0 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 16) and to (ii) a value that represented the synapse state and an association rate constant of (1.5 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 121) were obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules observed. (D) (i) The dwell times in the abortive complex state for the DNA molecules containing parallel loxP sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [R2 = 0.98, τ1 = (4.0 ± 0.7) × 10−3 s−1, n = 7]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [R2 = 0.98, k1 = (6.8 ± 0.8) × 10−3 s−1, n = 148] or a bi-exponential decay algorithm [R2 = 1.00, k1 = (4.1 ± 1.9) × 10−2 s−1, A1 = 0.90; k2 = (2.6 ± 0.2) × 10−3 s−1, A2 = 0.10, n = 148]. Solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL. (E) The mechanism through which bacteriophage P1 CreK201A mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent Cre recombinases. The arrows on the DNA molecule indicate the parallel loxP sites.

Mentions: It has been known that the cleavage-deficient mutant CreK201A, from which the required general acid catalyst that activates O5' has been removed, possesses a similar synapsis ability as wild-type Cre, with Kd = 8.9 ± 0.6 nM (12). Therefore, CreK201A was used to perform the same experiments and analyses in order to verify whether cleavage is required for the formation of the stable synaptic complex in the Cre-loxP system. The BM time traces are shown in Figure 6A, and there are two typical reaction behaviors, similar to those of CreY324F. One reaction behavior is that a DNA molecule formed a synaptic complex, but there was no formation of a recombinant product verified by the SDS challenge, and the other is that a DNA molecule failed to synapse during the duration of the observations. Analysis of 59 molecules with a BM amplitude between 70 and 91 nm showed that 43 molecules exhibited a significant change in BM in response to the addition of CreK201A recombinase. The BM histograms are shown in Figure 6B, which are similar to those observed for CreY324F. Association rate constants of (2.0 ± 0.1) × 104 M−1 s−1 and (1.5 ± 0.1) × 104 M−1 s−1 for the formation of abortive complexes and synaptic complexes, respectively, were obtained by fitting the duration histograms to a single exponential model [Figure 6C(i–ii)]. The stability of the abortive complex was obtained by fitting the abortive complex duration histogram to a single exponential model with a dissociation rate constant of (4.0 ± 0.7) × 10−3 s−1 [Figure 6D(i)]. When the duration of the synaptic complex was analyzed further, it was found that the duration distribution was similar to that observed in the wild-type Cre-loxP system, and a bi-exponential decay model was used. The fast process, with a rate constant of (4.1 ± 1.9) × 10−2 s−1, represents the decomposition of the wayward complex. The same rate constants were obtained for both wild-type Cre and CreY324F, which reinforced the existence of a wayward complex, in the Cre-loxP system. On the other hand, it has been shown that the catalysis recombination process, i.e. strand cleavage/ligation, is reversible. Limited by the resolution of the TPM, the change in BM amplitude can only signal the decomposition of the synaptic complex, and the BM amplitudes observed for synaptic complexes, cleavage synaptic complexes and Holliday junction intermediates showed insignificant differences. Moreover, it has been reported that the rate-limiting step in the Cre-mediated recombination process is the dissociation of the recombined synapse (31). Therefore, the slow process observed in wild-type Cre and CreK201A, with a rate constant of (2.6 ± 0.2) × 10−3 s−1, represents the decomposition of the stable synaptic complex as well as the decomposition of the recombined synaptic complex.Figure 6.


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 for 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreK201A. (A) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel loxP sites in response to the addition of CreK201A. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. (B) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 78.8 ± 10.2 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (61.0 ± 1.3 nm and 44.0 ± 12.0 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude at 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge at 30 min of incubation time (n = 43). (C) The distribution of the dwell times between recombinase addition and the change in the BM amplitude to (i) a value that represented the abortive complex state and an association rate constant of (2.0 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 16) and to (ii) a value that represented the synapse state and an association rate constant of (1.5 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 121) were obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules observed. (D) (i) The dwell times in the abortive complex state for the DNA molecules containing parallel loxP sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [R2 = 0.98, τ1 = (4.0 ± 0.7) × 10−3 s−1, n = 7]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [R2 = 0.98, k1 = (6.8 ± 0.8) × 10−3 s−1, n = 148] or a bi-exponential decay algorithm [R2 = 1.00, k1 = (4.1 ± 1.9) × 10−2 s−1, A1 = 0.90; k2 = (2.6 ± 0.2) × 10−3 s−1, A2 = 0.10, n = 148]. Solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL. (E) The mechanism through which bacteriophage P1 CreK201A mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent Cre recombinases. The arrows on the DNA molecule indicate the parallel loxP sites.
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Related In: Results  -  Collection

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gks274-F6: Complex formation and recombination events for 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreK201A. (A) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel loxP sites in response to the addition of CreK201A. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. (B) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 78.8 ± 10.2 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (61.0 ± 1.3 nm and 44.0 ± 12.0 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude at 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge at 30 min of incubation time (n = 43). (C) The distribution of the dwell times between recombinase addition and the change in the BM amplitude to (i) a value that represented the abortive complex state and an association rate constant of (2.0 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 16) and to (ii) a value that represented the synapse state and an association rate constant of (1.5 ± 0.1) × 104 M−1 s−1 (R2 = 0.98, n = 121) were obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules observed. (D) (i) The dwell times in the abortive complex state for the DNA molecules containing parallel loxP sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [R2 = 0.98, τ1 = (4.0 ± 0.7) × 10−3 s−1, n = 7]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [R2 = 0.98, k1 = (6.8 ± 0.8) × 10−3 s−1, n = 148] or a bi-exponential decay algorithm [R2 = 1.00, k1 = (4.1 ± 1.9) × 10−2 s−1, A1 = 0.90; k2 = (2.6 ± 0.2) × 10−3 s−1, A2 = 0.10, n = 148]. Solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL. (E) The mechanism through which bacteriophage P1 CreK201A mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel loxP sites. The empty circles represent Cre recombinases. The arrows on the DNA molecule indicate the parallel loxP sites.
Mentions: It has been known that the cleavage-deficient mutant CreK201A, from which the required general acid catalyst that activates O5' has been removed, possesses a similar synapsis ability as wild-type Cre, with Kd = 8.9 ± 0.6 nM (12). Therefore, CreK201A was used to perform the same experiments and analyses in order to verify whether cleavage is required for the formation of the stable synaptic complex in the Cre-loxP system. The BM time traces are shown in Figure 6A, and there are two typical reaction behaviors, similar to those of CreY324F. One reaction behavior is that a DNA molecule formed a synaptic complex, but there was no formation of a recombinant product verified by the SDS challenge, and the other is that a DNA molecule failed to synapse during the duration of the observations. Analysis of 59 molecules with a BM amplitude between 70 and 91 nm showed that 43 molecules exhibited a significant change in BM in response to the addition of CreK201A recombinase. The BM histograms are shown in Figure 6B, which are similar to those observed for CreY324F. Association rate constants of (2.0 ± 0.1) × 104 M−1 s−1 and (1.5 ± 0.1) × 104 M−1 s−1 for the formation of abortive complexes and synaptic complexes, respectively, were obtained by fitting the duration histograms to a single exponential model [Figure 6C(i–ii)]. The stability of the abortive complex was obtained by fitting the abortive complex duration histogram to a single exponential model with a dissociation rate constant of (4.0 ± 0.7) × 10−3 s−1 [Figure 6D(i)]. When the duration of the synaptic complex was analyzed further, it was found that the duration distribution was similar to that observed in the wild-type Cre-loxP system, and a bi-exponential decay model was used. The fast process, with a rate constant of (4.1 ± 1.9) × 10−2 s−1, represents the decomposition of the wayward complex. The same rate constants were obtained for both wild-type Cre and CreY324F, which reinforced the existence of a wayward complex, in the Cre-loxP system. On the other hand, it has been shown that the catalysis recombination process, i.e. strand cleavage/ligation, is reversible. Limited by the resolution of the TPM, the change in BM amplitude can only signal the decomposition of the synaptic complex, and the BM amplitudes observed for synaptic complexes, cleavage synaptic complexes and Holliday junction intermediates showed insignificant differences. Moreover, it has been reported that the rate-limiting step in the Cre-mediated recombination process is the dissociation of the recombined synapse (31). Therefore, the slow process observed in wild-type Cre and CreK201A, with a rate constant of (2.6 ± 0.2) × 10−3 s−1, represents the decomposition of the stable synaptic complex as well as the decomposition of the recombined synaptic complex.Figure 6.

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