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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 of 1267 bp DNA molecules containing inverse loxP sites. (A) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing inverse loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed and then returned to the original substrate, corresponding to either no reaction or completion of recombination. (ii) An example of a molecule that synapsed and was then trapped in a stable Holliday junction intermediate. (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 indicates the average BM value of the expected excision recombinant product. (B) Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing inverse loxP sites. The starting substrates are 1267 bp DNA molecules containing inverse loxP sites and exhibit an average BM amplitude of 79.8 ± 7.5 nm. Two complexes are formed after interaction with Cre recombinase. The first complex is the synaptic complex, which corresponds to the formation of a Cre tetramer with an average BM value of 48.6 ± 10.2 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations and corresponds to the association of Cre dimers with loxP sites with an average BM value of 64.3 ± 3.6 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 either failed to complete recombination or formed the recombinant product after completion of the recombination process; (ii) a molecule that synapsed but was trapped in a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (C) (i) The distribution of the BM amplitude before the addition of Cre recombinase showed an average value of 79.8 ± 7.5 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (64.3 ± 3.6, 48.6 ± 10.2 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 = 101). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (D) to a value representing the abortive complex state and an association rate constant of (8.1 ± 0.5) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 18) were obtained. (E) To a value that represents the synapse state, and an association rate constant of (6.4 ± 0.3) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 81) 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.
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gks274-F3: Complex formation and recombination events of 1267 bp DNA molecules containing inverse loxP sites. (A) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing inverse loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed and then returned to the original substrate, corresponding to either no reaction or completion of recombination. (ii) An example of a molecule that synapsed and was then trapped in a stable Holliday junction intermediate. (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 indicates the average BM value of the expected excision recombinant product. (B) Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing inverse loxP sites. The starting substrates are 1267 bp DNA molecules containing inverse loxP sites and exhibit an average BM amplitude of 79.8 ± 7.5 nm. Two complexes are formed after interaction with Cre recombinase. The first complex is the synaptic complex, which corresponds to the formation of a Cre tetramer with an average BM value of 48.6 ± 10.2 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations and corresponds to the association of Cre dimers with loxP sites with an average BM value of 64.3 ± 3.6 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 either failed to complete recombination or formed the recombinant product after completion of the recombination process; (ii) a molecule that synapsed but was trapped in a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (C) (i) The distribution of the BM amplitude before the addition of Cre recombinase showed an average value of 79.8 ± 7.5 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (64.3 ± 3.6, 48.6 ± 10.2 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 = 101). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (D) to a value representing the abortive complex state and an association rate constant of (8.1 ± 0.5) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 18) were obtained. (E) To a value that represents the synapse state, and an association rate constant of (6.4 ± 0.3) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 81) 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.

Mentions: To distinguish whether Holliday junction intermediates or excision products are formed within the 30-min incubation time, the same experiments were performed on 1267 bp DNA molecules containing inverse loxP sites separated by 653 bp. The recombination process in the DNA molecules containing inverse loxP sites results in an inverted 653 bp DNA segment between the two sites and an unchanged DNA length for the recombinant product. Examples of single-molecule time traces are shown in Figure 3A(i)–(iii). For molecules that have proceeded to the synapse state, there are two possible outcomes following an SDS challenge: first, a return to the original DNA substrate or the formation of an inverse recombinant product [Figure 3B(i)], and second, remaining as a shortened DNA product, representing the stable Holliday junction intermediate [Figure 3B(ii)]. Molecules that are trapped at the abortive complex will return to the original DNA substrate prior to the SDS challenge. Of the 301 analyzed molecules with a starting BM value of 79.8 ± 7.5 nm, 101 molecules exhibited a change in their BM amplitude in response to the addition of Cre recombinase. Of the 101 analyzed molecules, 18 showed a decrease in BM amplitude to an average value of 64.3 ± 3.6 nm within the 95% CL and failed to synapse within the experimental duration [Figure 3A(iii) and 3C(ii)], indicating the formation of abortive complexes. When the remaining analyzed molecules were examined, the BM amplitude was found to decrease rapidly to an average value of 48.6 ± 10.2 nm within the 95% CL, indicating the formation of synaptic complexes [Figure 3C(ii)]. The BM amplitude of the synapse state for the DNA molecules containing inverse loxP sites is different from that of DNA molecules containing parallel loxP sites. The discrepancy in the BM amplitude between the synapse states occurs because the two loxP sites, when in the correct synapse state, are expected to be in an anti-parallel orientation. This constraint results in extra topological bending of DNA molecules containing inverse loxP sites (13,16,40). After 30 min of incubation with Cre recombinase, the percentage of DNA molecules in the abortive complex decreased because they were returning to the original DNA substrate state [Figure 3C(iii)]. Immediately upon the SDS challenge at 30 min reaction time, the BM amplitude of most of the DNA molecules (90%) remained at a low value of 48.6 ± 10.2 nm, indicating the formation of stable Holliday junction intermediates. Similar association constants of (8.1 ± 0.5) × 104 Mβˆ’1 sβˆ’1 and (6.4 ± 0.3) × 104 Mβˆ’1 sβˆ’1 for the abortive complexes and synaptic complexes, respectively, were calculated by fitting the dwell histograms to a single exponential model (Figure 3D and E).Figure 3.


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 inverse loxP sites. (A) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing inverse loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed and then returned to the original substrate, corresponding to either no reaction or completion of recombination. (ii) An example of a molecule that synapsed and was then trapped in a stable Holliday junction intermediate. (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 indicates the average BM value of the expected excision recombinant product. (B) Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing inverse loxP sites. The starting substrates are 1267 bp DNA molecules containing inverse loxP sites and exhibit an average BM amplitude of 79.8 ± 7.5 nm. Two complexes are formed after interaction with Cre recombinase. The first complex is the synaptic complex, which corresponds to the formation of a Cre tetramer with an average BM value of 48.6 ± 10.2 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations and corresponds to the association of Cre dimers with loxP sites with an average BM value of 64.3 ± 3.6 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 either failed to complete recombination or formed the recombinant product after completion of the recombination process; (ii) a molecule that synapsed but was trapped in a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (C) (i) The distribution of the BM amplitude before the addition of Cre recombinase showed an average value of 79.8 ± 7.5 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (64.3 ± 3.6, 48.6 ± 10.2 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 = 101). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (D) to a value representing the abortive complex state and an association rate constant of (8.1 ± 0.5) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 18) were obtained. (E) To a value that represents the synapse state, and an association rate constant of (6.4 ± 0.3) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 81) 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.
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gks274-F3: Complex formation and recombination events of 1267 bp DNA molecules containing inverse loxP sites. (A) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing inverse loxP sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed and then returned to the original substrate, corresponding to either no reaction or completion of recombination. (ii) An example of a molecule that synapsed and was then trapped in a stable Holliday junction intermediate. (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 indicates the average BM value of the expected excision recombinant product. (B) Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing inverse loxP sites. The starting substrates are 1267 bp DNA molecules containing inverse loxP sites and exhibit an average BM amplitude of 79.8 ± 7.5 nm. Two complexes are formed after interaction with Cre recombinase. The first complex is the synaptic complex, which corresponds to the formation of a Cre tetramer with an average BM value of 48.6 ± 10.2 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations and corresponds to the association of Cre dimers with loxP sites with an average BM value of 64.3 ± 3.6 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 either failed to complete recombination or formed the recombinant product after completion of the recombination process; (ii) a molecule that synapsed but was trapped in a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. (C) (i) The distribution of the BM amplitude before the addition of Cre recombinase showed an average value of 79.8 ± 7.5 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (64.3 ± 3.6, 48.6 ± 10.2 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 = 101). The distribution of the dwell times between recombinase addition and the change in the BM amplitude (D) to a value representing the abortive complex state and an association rate constant of (8.1 ± 0.5) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 18) were obtained. (E) To a value that represents the synapse state, and an association rate constant of (6.4 ± 0.3) × 104 Mβˆ’1 sβˆ’1 (R2 = 0.98, n = 81) 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.
Mentions: To distinguish whether Holliday junction intermediates or excision products are formed within the 30-min incubation time, the same experiments were performed on 1267 bp DNA molecules containing inverse loxP sites separated by 653 bp. The recombination process in the DNA molecules containing inverse loxP sites results in an inverted 653 bp DNA segment between the two sites and an unchanged DNA length for the recombinant product. Examples of single-molecule time traces are shown in Figure 3A(i)–(iii). For molecules that have proceeded to the synapse state, there are two possible outcomes following an SDS challenge: first, a return to the original DNA substrate or the formation of an inverse recombinant product [Figure 3B(i)], and second, remaining as a shortened DNA product, representing the stable Holliday junction intermediate [Figure 3B(ii)]. Molecules that are trapped at the abortive complex will return to the original DNA substrate prior to the SDS challenge. Of the 301 analyzed molecules with a starting BM value of 79.8 ± 7.5 nm, 101 molecules exhibited a change in their BM amplitude in response to the addition of Cre recombinase. Of the 101 analyzed molecules, 18 showed a decrease in BM amplitude to an average value of 64.3 ± 3.6 nm within the 95% CL and failed to synapse within the experimental duration [Figure 3A(iii) and 3C(ii)], indicating the formation of abortive complexes. When the remaining analyzed molecules were examined, the BM amplitude was found to decrease rapidly to an average value of 48.6 ± 10.2 nm within the 95% CL, indicating the formation of synaptic complexes [Figure 3C(ii)]. The BM amplitude of the synapse state for the DNA molecules containing inverse loxP sites is different from that of DNA molecules containing parallel loxP sites. The discrepancy in the BM amplitude between the synapse states occurs because the two loxP sites, when in the correct synapse state, are expected to be in an anti-parallel orientation. This constraint results in extra topological bending of DNA molecules containing inverse loxP sites (13,16,40). After 30 min of incubation with Cre recombinase, the percentage of DNA molecules in the abortive complex decreased because they were returning to the original DNA substrate state [Figure 3C(iii)]. Immediately upon the SDS challenge at 30 min reaction time, the BM amplitude of most of the DNA molecules (90%) remained at a low value of 48.6 ± 10.2 nm, indicating the formation of stable Holliday junction intermediates. Similar association constants of (8.1 ± 0.5) × 104 Mβˆ’1 sβˆ’1 and (6.4 ± 0.3) × 104 Mβˆ’1 sβˆ’1 for the abortive complexes and synaptic complexes, respectively, were calculated by fitting the dwell histograms to a single exponential model (Figure 3D and E).Figure 3.

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