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Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

Fan HF, Cheng YS, Ma CH, Jayaram M - Nucleic Acids Res. (2015)

Bottom Line: These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes.The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps.The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.

View Article: PubMed Central - PubMed

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

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Analysis of Cre(W315F) and Flpe(W330F) by TPM. (A, B) The histogram plots were obtained from a 30 min time course assay. (C–F) For deriving kinetic constants, the dwell time histograms in C, D and F and those in the top panel of E (non-productive complexes formed by Cre(W315F)) were fitted to a single exponential model. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(W315F)) fitted to a single exponential model (solid curve) gave k1 = (4.3 ± 0.5) x 10−3 s−1 (R2 = 0.93). The same data fitted to a double exponential model (dashed curve) to account for reversal of the Holliday junction intermediate (as noted under Figure 4) gave k1′ = (1.6 ± 0.33) x 10−2 s−1 and k2′ = (1.8 ± 0.2) x 10−3 s−1 (R2 = 0.99) for the decay of the wayward complexes and recombinogenic complexes (by reversal of Holliday junction formation), respectively. The derived kinetic constants are summarized in Tables 2 and 3. Not included in the tables are kNPf = (3.6 ± 0.1) x 104 M−1 s−1 and kNPd = (4.0 ± 0.3) x 10−2 s−1 for Cre(W315F); kNPf = (2.0 ± 0.1) x 104 M−1 s−1 and kNPd = (2.7 ± 0.3) x 10−2 M−1 s−1 for Flpe(W330F).
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Figure 6: Analysis of Cre(W315F) and Flpe(W330F) by TPM. (A, B) The histogram plots were obtained from a 30 min time course assay. (C–F) For deriving kinetic constants, the dwell time histograms in C, D and F and those in the top panel of E (non-productive complexes formed by Cre(W315F)) were fitted to a single exponential model. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(W315F)) fitted to a single exponential model (solid curve) gave k1 = (4.3 ± 0.5) x 10−3 s−1 (R2 = 0.93). The same data fitted to a double exponential model (dashed curve) to account for reversal of the Holliday junction intermediate (as noted under Figure 4) gave k1′ = (1.6 ± 0.33) x 10−2 s−1 and k2′ = (1.8 ± 0.2) x 10−3 s−1 (R2 = 0.99) for the decay of the wayward complexes and recombinogenic complexes (by reversal of Holliday junction formation), respectively. The derived kinetic constants are summarized in Tables 2 and 3. Not included in the tables are kNPf = (3.6 ± 0.1) x 104 M−1 s−1 and kNPd = (4.0 ± 0.3) x 10−2 s−1 for Cre(W315F); kNPf = (2.0 ± 0.1) x 104 M−1 s−1 and kNPd = (2.7 ± 0.3) x 10−2 M−1 s−1 for Flpe(W330F).

Mentions: The kinetic constants for Cre and Cre(K201A) were determined in previous work (24). The values for all other proteins were determined from time traces of individual molecules represented in the data shown in Figures 3–6. The rate constants for recombination (column 6) by Cre(H289A) and Cre(W315F) were obtained from the data in panel E of Figures 4 and 6, respectively, by fitting them to a double exponential model (Materials and Methods). An independent estimate of the rate constant for recombination by Cre(W315F) using a DNA substrate containing head-to-head loxP sites gave a value close to that shown here (Supplementary Figure S4). kPSf = rate constant for the formation of pre-synaptic complexes; kRSf = rate constant for the formation of recombination-competent synaptic complexes; kWWf = rate constant for the formation of wayward synaptic complexes; k*WWf = rate constant for the formation of long-lived wayward complexes assembled by Cre(K201A); kWWd = rate constant for the decomposition of wayward complexes; k*WWd = rate constant for the decomposition of long-lived wayward complexes formed by Cre(K201A); kREC = rate constant for recombination (conversion of synaptic complexes into recombinant products).


Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

Fan HF, Cheng YS, Ma CH, Jayaram M - Nucleic Acids Res. (2015)

Analysis of Cre(W315F) and Flpe(W330F) by TPM. (A, B) The histogram plots were obtained from a 30 min time course assay. (C–F) For deriving kinetic constants, the dwell time histograms in C, D and F and those in the top panel of E (non-productive complexes formed by Cre(W315F)) were fitted to a single exponential model. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(W315F)) fitted to a single exponential model (solid curve) gave k1 = (4.3 ± 0.5) x 10−3 s−1 (R2 = 0.93). The same data fitted to a double exponential model (dashed curve) to account for reversal of the Holliday junction intermediate (as noted under Figure 4) gave k1′ = (1.6 ± 0.33) x 10−2 s−1 and k2′ = (1.8 ± 0.2) x 10−3 s−1 (R2 = 0.99) for the decay of the wayward complexes and recombinogenic complexes (by reversal of Holliday junction formation), respectively. The derived kinetic constants are summarized in Tables 2 and 3. Not included in the tables are kNPf = (3.6 ± 0.1) x 104 M−1 s−1 and kNPd = (4.0 ± 0.3) x 10−2 s−1 for Cre(W315F); kNPf = (2.0 ± 0.1) x 104 M−1 s−1 and kNPd = (2.7 ± 0.3) x 10−2 M−1 s−1 for Flpe(W330F).
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Figure 6: Analysis of Cre(W315F) and Flpe(W330F) by TPM. (A, B) The histogram plots were obtained from a 30 min time course assay. (C–F) For deriving kinetic constants, the dwell time histograms in C, D and F and those in the top panel of E (non-productive complexes formed by Cre(W315F)) were fitted to a single exponential model. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(W315F)) fitted to a single exponential model (solid curve) gave k1 = (4.3 ± 0.5) x 10−3 s−1 (R2 = 0.93). The same data fitted to a double exponential model (dashed curve) to account for reversal of the Holliday junction intermediate (as noted under Figure 4) gave k1′ = (1.6 ± 0.33) x 10−2 s−1 and k2′ = (1.8 ± 0.2) x 10−3 s−1 (R2 = 0.99) for the decay of the wayward complexes and recombinogenic complexes (by reversal of Holliday junction formation), respectively. The derived kinetic constants are summarized in Tables 2 and 3. Not included in the tables are kNPf = (3.6 ± 0.1) x 104 M−1 s−1 and kNPd = (4.0 ± 0.3) x 10−2 s−1 for Cre(W315F); kNPf = (2.0 ± 0.1) x 104 M−1 s−1 and kNPd = (2.7 ± 0.3) x 10−2 M−1 s−1 for Flpe(W330F).
Mentions: The kinetic constants for Cre and Cre(K201A) were determined in previous work (24). The values for all other proteins were determined from time traces of individual molecules represented in the data shown in Figures 3–6. The rate constants for recombination (column 6) by Cre(H289A) and Cre(W315F) were obtained from the data in panel E of Figures 4 and 6, respectively, by fitting them to a double exponential model (Materials and Methods). An independent estimate of the rate constant for recombination by Cre(W315F) using a DNA substrate containing head-to-head loxP sites gave a value close to that shown here (Supplementary Figure S4). kPSf = rate constant for the formation of pre-synaptic complexes; kRSf = rate constant for the formation of recombination-competent synaptic complexes; kWWf = rate constant for the formation of wayward synaptic complexes; k*WWf = rate constant for the formation of long-lived wayward complexes assembled by Cre(K201A); kWWd = rate constant for the decomposition of wayward complexes; k*WWd = rate constant for the decomposition of long-lived wayward complexes formed by Cre(K201A); kREC = rate constant for recombination (conversion of synaptic complexes into recombinant products).

Bottom Line: These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes.The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps.The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus