Synapsis stimulates two of the four Cre subunits for cleavage of loxP (Ghosh et al., 2005a).
The protein:protein interactions formed in this tetrameric complex likely facilitate the cleavage activity (Ghosh et al., 2007), but it is not known if these contacts are required for Cre to cleave
DNA.
The cleavage activity of the Cre monomer bound to loxP half site was studied by Peng Yuan,
a former graduate student. At high concentrations (4uM complex), Cre was capable of cleaving the half site, trapping approximately 50% of Cre in a covalent intermediate (Yuan, 2008). Although Cre was capable of this cleavage in solution, no evidence for covalent intermediate formation was observed in the crystal structure. At such high concentrations, extensive
protein:protein interactions are likely formed, thereby activating Cre for cleavage. The cleavage activity was measured as a function of Cre-loxP complex concentration. Cleavage increased with
complex concentration, indicating that the observed cleavage was likely due to protein:protein interactions, and not to the Cre monomer acting on an isolated loxP substrate. Additional
experiments with a Cre mutant (A36V) that is known to be partially defective in synapsis (Hoess
et al., 1987; Wierzbicki et al., 1987), further supported the conclusion that Cre requires
intersubunit interactions for cleavage.
The dimeric Cre-loxP complex might be better suited to have cleavage activity considering
the Cre dimer forms protein:protein interactions, and hN from one or both subunits will be able to bind the hydrophobic pocket of the partner subunit. The formation of synapsis has been shown to accelerate cleavage (Ghosh et al., 2005a; Guo et al., 1999; Hoess, R H, Wierzbicki A, Abremski
KE, 1990), so in order to be able to determine if Cre is cleaving as a dimer, the reaction must be performed under conditions where synapsis does not occur. Assuming Cre is at saturating concentrations, the KD of synapsis of Cre with loxP is approximately 10nM (Ghosh et al., 2007).
concentrations of substrate nearly 100 fold below the KD of synapsis, and found that cleavage of
the bottom strand (GC half site) approaches zero, but the cleavage of the top strand (AT half site) is robust even at the lowest substrate concentrations (Ghosh et al., 2005a). The results suggest
that the Cre dimer is capable of cleaving the top strand of loxP, but cleavage of the bottom strand
appears to require synapsis. This set of experiments was repeated with Cre A36V, a synapsis defective mutant, to determine if a low level of synapsis is responsible for the previously measured cleavage activity (Fig. 5.4). Wildtype Cre robustly cleaved the top strand of the phosphorothiolate substrate. Surprisingly, cleavage of the bottom strand does not go to zero, but instead reaches a plateau of about 12% at concentrations 1000 fold below the KD for synapsis.
Interestingly, A36V displays a steady basal level of cleavage for almost the entire concentration range tested, which supports the idea that the Cre dimer is active for cleavage. However, the phosphorothiolate substrate is an unnatural substrate that alleviates the need for a general acid to assist the leaving group during cleavage, therefore the requirements to cleave
phosphorothiolate DNA are less stringent than those for a natural substrate.
To eliminate the possibility of the that cleavage by the Cre-loxP dimer is an artifact of the
artificial phosphorothiolate substrates, cleavage was measured with a suicide substrate that contained a natural phosphodiester linkage at the scissile phosphate. Cleavage was assayed at a concentration of loxP ranging from 2pM to 2000 pM. The lowest concentration was 5000 fold
below the KD of synapsis (Ghosh et al., 2007). Reactions with wild type Cre were incubated for 1
hour at 37 °C. At the lowest concentration, cleavage was measured to be 10% of total counts and remained nearly constant over a sixty fold increase in substrate concentration before
increasing significantly at concentrations close to the KD of synapsis. The trend was the same for
cleavage of both top and bottom strands. The increase in cleavage near the end of the measured concentration range of the assay is consistent with stimulation from the formation of synapsis. The concentration dependence of synapsis in Cre has been studied and modeled (Ghosh et al., 2007). Using this model, the approximate amount of synapsis formed can be calculated for the concentrations of substrate used in the cleavage experiment (Fig. 5.4). At 2pM substrate, less
than 0.03% Cre bound loxP is in a synaptic complex, and while this amount will increase with concentration, the cleavage product does not. This indicates that the observed cleavage is independent of synapsis suggesting that observed cleavage is from the Cre-loxP dimer complex
(Fig. 5.5).
The ability of the dimeric Cre-loxP complex to cleave but not the Cre monomer to cleave
DNA substrate suggests that intersubunit protein contacts must be made to activate Cre for cleavage. It is unclear, however, if one or both Cre subunits in the dimer are capable of cleavage. Although the use of nicked suicide substrates allows cleavage to be measured at a normal phosphodiester linkage, the presence of the nick may influence bending of the loxP site, which is
known to be important in regulating the activity of Cre in the synaptic complex (Ghosh et al.,
2005a; Guo et al., 1997; Guo et al., 1999). Without structural information for the Cre-dimer, it is
Figure 5.4
WT Cre cleaves the TS of loxP preferentially as a dimer.
Cre cleavage as a function of loxP concentration for wildtype (A) and A36V (B). Use of a suicide phosphorothiolate substrate was used to trap Cre-loxP in a covalent complex. Reactions were separated by SDS-PAGE after 60 mins. Concentrations of substrate used were, 6, 22, 66, 200, 2000, 20,000, and 100,000(A36V only) pM A. Bottom strand product (BS) runs with slower mobilty than top strand covalent product (TS). Graphs on bottom show fraction cleaved vs. fraction synapsed. The fraction synapsed was calculated using a published model for Cre (Ghosh, Guo & Van Duyne, 2007). The x-axis is plotted on a log scale to show the entire concentration range clearly.