PaqCI

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1 to 7 of 7 Results

Figure 9 DNA pre-bound at all four sites in the PaqCI tetramer is cut sequentially Feb 3, 2023 Kennedy, Madison, 2023, "Figure 9 DNA pre-bound at all four sites in the PaqCI tetramer is cut sequentially", https://doi.org/10.7910/DVN/2SZXFW, Harvard Dataverse, V1 Panel a. Cleavage of a four-site plasmid substrate either pre-bound or not pre-bound with a 1:1 enzyme-binding-site to DNA-sites ratio. The pre-bound reaction contained 1 ug substrate DNA (37 nM sites) and 4.3 units (37 nM) of PaqCI per ug DNA per 50 ul rCutSmart buffer lacking Mg++ ions and enzyme was allowed to bind for 15 minutes at 37C. An aliq...
Figure 8 Time-dependent digestion of plasmids with four PaqCI target sites indicate a sequential cleavage at individual sites Feb 3, 2023 Kennedy, Madison, 2023, "Figure 8 Time-dependent digestion of plasmids with four PaqCI target sites indicate a sequential cleavage at individual sites", https://doi.org/10.7910/DVN/QEEZ7Y, Harvard Dataverse, V1 Panel a. Cleavage of a head-to-head four-site substrate with and without an activator added in trans. The no activator reaction contained 1 ug substrate DNA (37 nM sites) and 2 units (17.2 nM) of PaqCI per ug DNA in 50 ul rCutSmart buffer at 37C. The activator reaction had the same substrate and PaqCI enzyme together with 80 nM activating oligodupl...
Figure 1 Biochemical analyses of PaqCI cleavage activity Oct 13, 2022 Kennedy, Madison, 2022, "Figure 1 Biochemical analyses of PaqCI cleavage activity", https://doi.org/10.7910/DVN/TIHGAY, Harvard Dataverse, V1 Panel a: PaqCI demonstrates substrate turnover when acting on a stoichiometric excess of DNA targets. Digest time points are quenched at 10 minutes and 60 minutes with protein concentration ranging from 0.3-8.0 units with the DNA concentration held at 200 nM in each vial. In the 10 minute digest, complete cleavage of the oligo is only seen in conce...
Figure 2 Time-dependent digestion of plasmids with two PaqCI target sites indicate a sequential cleavage profile Oct 13, 2022 Kennedy, Madison, 2022, "Figure 2 Time-dependent digestion of plasmids with two PaqCI target sites indicate a sequential cleavage profile", https://doi.org/10.7910/DVN/62YXDO, Harvard Dataverse, V1 The reaction contained 1 ug substrate DNA and 2 units/ug PaqCI endonuclease in rCutSmart buffer at 37C. Digest time points were quenched at 0.25 min, 0.5 min, 1 min, 3 min, 5 min, 10 min, 30 min, and 60 minutes with 1 ug dual site substrate and 2 units/ug PaqCI as indicated above each lane. The mobilities of the super coil (SC) and the cleaved line...
Figure S5 Plasmids with two PaqCI target sites show potential sequential cleavage when trans oligo enhancer is added Oct 13, 2022 Kennedy, Madison, 2022, "Figure S5 Plasmids with two PaqCI target sites show potential sequential cleavage when trans oligo enhancer is added", https://doi.org/10.7910/DVN/XTQZXI, Harvard Dataverse, V1 The reaction contained 1 ug substrate DNA, 2 units/ug PaqCI endonuclease and 40 nM/unit activating oligonucleotide in rCutSmart buffer at 37C. Reactions were quenched at the time points indicated above each lane. The mobilities of the super coil (SC) and the linear (Lin) forms of the plasmid are indicated in the middle. On the right of the gels is...
Figure S3 CryoEM analysis of PaqCI DNA-bound enzyme, Panel a Oct 13, 2022 Kennedy, Madison, 2022, "Figure S3 CryoEM analysis of PaqCI DNA-bound enzyme, Panel a", https://doi.org/10.7910/DVN/5GHTIV, Harvard Dataverse, V1 Size exclusion chromatographic elution behavior of PaqCI enzyme in the presence of a stoichiometric excess of double strand DNA (see Figure 3a) indicates formation of a stable DNA-bound enzyme complex. The elution profiles for free protein, free DNA and a 1:1 stoichiometric mixture of protein + DNA are shown as bold colored lines; the elution of mo...
Figure S1 Purification and solution behavior of PaqCI Oct 13, 2022 Kennedy, Madison, 2022, "Figure S1 Purification and solution behavior of PaqCI", https://doi.org/10.7910/DVN/G8XG68, Harvard Dataverse, V1 Panel a: SDS-PAGE gel of induced cell lysate and purification via metal affinity chromatography. Panel b: Size exclusion chromatographic elution of final purified PaqCI used for subsequent structural analyses. The protein elution profile is dark blue; underlying elution of molecular weight standards is light dashed grey. The expected peak elution v...

Figure 9 DNA pre-bound at all four sites in the PaqCI tetramer is cut sequentially

Feb 3, 2023

Kennedy, Madison, 2023, "Figure 9 DNA pre-bound at all four sites in the PaqCI tetramer is cut sequentially", https://doi.org/10.7910/DVN/2SZXFW, Harvard Dataverse, V1

Panel a. Cleavage of a four-site plasmid substrate either pre-bound or not pre-bound with a 1:1 enzyme-binding-site to DNA-sites ratio. The pre-bound reaction contained 1 ug substrate DNA (37 nM sites) and 4.3 units (37 nM) of PaqCI per ug DNA per 50 ul rCutSmart buffer lacking Mg++ ions and enzyme was allowed to bind for 15 minutes at 37C. An aliq...

Figure 8 Time-dependent digestion of plasmids with four PaqCI target sites indicate a sequential cleavage at individual sites

Feb 3, 2023

Kennedy, Madison, 2023, "Figure 8 Time-dependent digestion of plasmids with four PaqCI target sites indicate a sequential cleavage at individual sites", https://doi.org/10.7910/DVN/QEEZ7Y, Harvard Dataverse, V1

Panel a. Cleavage of a head-to-head four-site substrate with and without an activator added in trans. The no activator reaction contained 1 ug substrate DNA (37 nM sites) and 2 units (17.2 nM) of PaqCI per ug DNA in 50 ul rCutSmart buffer at 37C. The activator reaction had the same substrate and PaqCI enzyme together with 80 nM activating oligodupl...

Figure 1 Biochemical analyses of PaqCI cleavage activity

Oct 13, 2022

Kennedy, Madison, 2022, "Figure 1 Biochemical analyses of PaqCI cleavage activity", https://doi.org/10.7910/DVN/TIHGAY, Harvard Dataverse, V1

Panel a: PaqCI demonstrates substrate turnover when acting on a stoichiometric excess of DNA targets. Digest time points are quenched at 10 minutes and 60 minutes with protein concentration ranging from 0.3-8.0 units with the DNA concentration held at 200 nM in each vial. In the 10 minute digest, complete cleavage of the oligo is only seen in conce...

Figure 2 Time-dependent digestion of plasmids with two PaqCI target sites indicate a sequential cleavage profile

Oct 13, 2022

Kennedy, Madison, 2022, "Figure 2 Time-dependent digestion of plasmids with two PaqCI target sites indicate a sequential cleavage profile", https://doi.org/10.7910/DVN/62YXDO, Harvard Dataverse, V1

The reaction contained 1 ug substrate DNA and 2 units/ug PaqCI endonuclease in rCutSmart buffer at 37C. Digest time points were quenched at 0.25 min, 0.5 min, 1 min, 3 min, 5 min, 10 min, 30 min, and 60 minutes with 1 ug dual site substrate and 2 units/ug PaqCI as indicated above each lane. The mobilities of the super coil (SC) and the cleaved line...

Figure S5 Plasmids with two PaqCI target sites show potential sequential cleavage when trans oligo enhancer is added

Oct 13, 2022

Kennedy, Madison, 2022, "Figure S5 Plasmids with two PaqCI target sites show potential sequential cleavage when trans oligo enhancer is added", https://doi.org/10.7910/DVN/XTQZXI, Harvard Dataverse, V1

The reaction contained 1 ug substrate DNA, 2 units/ug PaqCI endonuclease and 40 nM/unit activating oligonucleotide in rCutSmart buffer at 37C. Reactions were quenched at the time points indicated above each lane. The mobilities of the super coil (SC) and the linear (Lin) forms of the plasmid are indicated in the middle. On the right of the gels is...

Figure S3 CryoEM analysis of PaqCI DNA-bound enzyme, Panel a

Oct 13, 2022

Kennedy, Madison, 2022, "Figure S3 CryoEM analysis of PaqCI DNA-bound enzyme, Panel a", https://doi.org/10.7910/DVN/5GHTIV, Harvard Dataverse, V1

Size exclusion chromatographic elution behavior of PaqCI enzyme in the presence of a stoichiometric excess of double strand DNA (see Figure 3a) indicates formation of a stable DNA-bound enzyme complex. The elution profiles for free protein, free DNA and a 1:1 stoichiometric mixture of protein + DNA are shown as bold colored lines; the elution of mo...

Figure S1 Purification and solution behavior of PaqCI

Oct 13, 2022

Kennedy, Madison, 2022, "Figure S1 Purification and solution behavior of PaqCI", https://doi.org/10.7910/DVN/G8XG68, Harvard Dataverse, V1

Panel a: SDS-PAGE gel of induced cell lysate and purification via metal affinity chromatography. Panel b: Size exclusion chromatographic elution of final purified PaqCI used for subsequent structural analyses. The protein elution profile is dark blue; underlying elution of molecular weight standards is light dashed grey. The expected peak elution v...

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