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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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Comparison between Cre(H289A) and Flpe(H305A) by TPM characterization. (A, B) The BM amplitude histograms represent the observation of molecules over a 30 min time course, followed immediately by SDS treatment. (C–F) The dwell time histograms in C, D and F, as well as those in the top panel of E (non-productive complexes formed by (Cre(H289A)), were fitted to a single exponential model to derive rate constants for formation or decay. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(H289A)) were fitted to either a single exponential model (solid curve) (R2 = 0.94; k1 = (6.3 ± 0.8) x 10−3 s−1) or to a double exponential model (dashed curve). The latter accounts for the decay of the wayward complexes (R2 = 0.99; k1′ = (2.0 ± 0.3) x 10−2 s−1) as well as that of recombinogenic complexes (R2 = 0.99; k2′ = (2.3 ± 0.3) x 10−3 s−1) behaving as pseudo-wayward complexes (due to reversal of Holliday junction formation). The estimated kinetic constants are summarized in Tables 2 and 3. Not included in the tables are: kNPf = (2.8 ± 0.1) x 104 M−1 s−1 and kNPd = (7.1 ± 0.9) x 10−3 s−1 for Cre(H289A); kNPf = (5.4 ± 0.8) x 104 M−1 s−1 and kNPd = (1.8 ± 0.1) x 10−2 s−1 for Flpe(H305A).
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Figure 4: Comparison between Cre(H289A) and Flpe(H305A) by TPM characterization. (A, B) The BM amplitude histograms represent the observation of molecules over a 30 min time course, followed immediately by SDS treatment. (C–F) The dwell time histograms in C, D and F, as well as those in the top panel of E (non-productive complexes formed by (Cre(H289A)), were fitted to a single exponential model to derive rate constants for formation or decay. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(H289A)) were fitted to either a single exponential model (solid curve) (R2 = 0.94; k1 = (6.3 ± 0.8) x 10−3 s−1) or to a double exponential model (dashed curve). The latter accounts for the decay of the wayward complexes (R2 = 0.99; k1′ = (2.0 ± 0.3) x 10−2 s−1) as well as that of recombinogenic complexes (R2 = 0.99; k2′ = (2.3 ± 0.3) x 10−3 s−1) behaving as pseudo-wayward complexes (due to reversal of Holliday junction formation). The estimated kinetic constants are summarized in Tables 2 and 3. Not included in the tables are: kNPf = (2.8 ± 0.1) x 104 M−1 s−1 and kNPd = (7.1 ± 0.9) x 10−3 s−1 for Cre(H289A); kNPf = (5.4 ± 0.8) x 104 M−1 s−1 and kNPd = (1.8 ± 0.1) x 10−2 s−1 for Flpe(H305A).

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)

Comparison between Cre(H289A) and Flpe(H305A) by TPM characterization. (A, B) The BM amplitude histograms represent the observation of molecules over a 30 min time course, followed immediately by SDS treatment. (C–F) The dwell time histograms in C, D and F, as well as those in the top panel of E (non-productive complexes formed by (Cre(H289A)), were fitted to a single exponential model to derive rate constants for formation or decay. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(H289A)) were fitted to either a single exponential model (solid curve) (R2 = 0.94; k1 = (6.3 ± 0.8) x 10−3 s−1) or to a double exponential model (dashed curve). The latter accounts for the decay of the wayward complexes (R2 = 0.99; k1′ = (2.0 ± 0.3) x 10−2 s−1) as well as that of recombinogenic complexes (R2 = 0.99; k2′ = (2.3 ± 0.3) x 10−3 s−1) behaving as pseudo-wayward complexes (due to reversal of Holliday junction formation). The estimated kinetic constants are summarized in Tables 2 and 3. Not included in the tables are: kNPf = (2.8 ± 0.1) x 104 M−1 s−1 and kNPd = (7.1 ± 0.9) x 10−3 s−1 for Cre(H289A); kNPf = (5.4 ± 0.8) x 104 M−1 s−1 and kNPd = (1.8 ± 0.1) x 10−2 s−1 for Flpe(H305A).
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Figure 4: Comparison between Cre(H289A) and Flpe(H305A) by TPM characterization. (A, B) The BM amplitude histograms represent the observation of molecules over a 30 min time course, followed immediately by SDS treatment. (C–F) The dwell time histograms in C, D and F, as well as those in the top panel of E (non-productive complexes formed by (Cre(H289A)), were fitted to a single exponential model to derive rate constants for formation or decay. The dwell time histograms in the bottom panel of E (synaptic complexes formed by Cre(H289A)) were fitted to either a single exponential model (solid curve) (R2 = 0.94; k1 = (6.3 ± 0.8) x 10−3 s−1) or to a double exponential model (dashed curve). The latter accounts for the decay of the wayward complexes (R2 = 0.99; k1′ = (2.0 ± 0.3) x 10−2 s−1) as well as that of recombinogenic complexes (R2 = 0.99; k2′ = (2.3 ± 0.3) x 10−3 s−1) behaving as pseudo-wayward complexes (due to reversal of Holliday junction formation). The estimated kinetic constants are summarized in Tables 2 and 3. Not included in the tables are: kNPf = (2.8 ± 0.1) x 104 M−1 s−1 and kNPd = (7.1 ± 0.9) x 10−3 s−1 for Cre(H289A); kNPf = (5.4 ± 0.8) x 104 M−1 s−1 and kNPd = (1.8 ± 0.1) x 10−2 s−1 for Flpe(H305A).
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