Frequently Asked Questions (FAQ)

Does this server support archiving or searching for homing endonucleases other than those from the LAGLIDADG family?

No, this resource is dedicated to archival and genomic target searches for LAGLIDADG homing endonucleases (LHEs), in order to support their continued development as tools for genome engineering and targeted gene modification.

How do I choose a name for my new LHE?

Naming conventions for LHEs (like all endonucleases) are dictated by rules established and described in Roberts et al. (2003) Nucleic Acids Research 31 (7): 1805 - 18172. We have listed the most important points for LHEs in links that you will find in the endonuclease entry forms, as well as under the 'Endonuclease entry help' menu items. Please do consult and abide by these nomenclature rules when entering new endonucleases.

Should my engineered variant of an already listed LHE be entered as a new engineered endonuclease, or should I edit the existing LHE through the 'Specificity Changing Mutations' Entry tool?

If your engineering consists solely of the identification of amino acid substitutions that alter DNA recognition specificity, we recommend simply adding those mutations to the existing wild-type LHE through the 'Specificity changing mutations' entry tool. If you have created and characterized an endonuclease that acts at a physiologically relevant genomic target (and, for example, have either validated its in vivo activity and/or examined its new specificity profile), then we recommend using the "Engineered endonuclease entry" tool" to create a novel entry.

I just noticed that I made a mistake in my entry, or I just noticed a mistake in an existing entry. What should I do?

Just use the 'Contact' link in the top menu and send the administrator an email describing the problem, and it will be corrected ASAP.

I just entered new information, but I don't see it yet in the browser. Why not?

New entries are temporarily quarantined for examination and approval by the webserver administrator. If you don't see your entry appear within 48 hours, please use the 'contact' tool and enquire about your entry.

Is a record kept of my genomic target searches?

No record of any sort is maintained of any searches. If you lose that information, please just rerun the search. All results are returned in real-time to your monitor.

What are PWM matrices, and how are they generated?

Position Weight Matrices (PWMs) provide simple representations of the fidelity of recognition and cleavage exhibited by LHEs at each position in their target site. Fidelity can range from being absolute for one base pair identity at a given position (corresponding to a value for 'Information Content' of 1.0 for a single base identity and 0.0 for the other three) to being absolutely nonspecific at a given position (corresponding to values of 0.25 for each of the four possible bases).

For details on the calculation of 'information content' across a protein-DNA recognition site, please refer to Schneider et al. (1986) "Information content of binding sites on nucleotide sequences" J. Mol. Biol. 188 (3): 415 - 431.

Entry of a PWM for a given LHE is based upon experimentally determined and validated specificity profile data for that endonuclease. Once a PWM has been entered for an LHE, that information is available for a PWM-based target search. Typically the data that yields a PWM corresponds to either (1) the relative ability of a given LHE to cleave target sites that harbor individual nucleotide sequence variants at each position, or (2) the relative frequency of nucleotide identities at each position calculated from the output of a selection experiment for cleavable target sites (usually from a partially randomized target site library).

Determination of specificity profiles via systematic measurements of relative cleavability can be done either using either in vitro digests with individual substrates or using yeast surface display combined with DNA staining and cleavage-dependent release, measured by flow cytometry.

For details of either method, see the following two references:

Thyme, S., Takeuchi, R., Jarjour, J., Scharenberg, A., Stoddard, B. L. and Baker, D. (2009) "Exploitation of homing endonuclease binding energy for catalysis and design" Nature 461: 1300 - 1304.

Jarjour, J., West-Foyle, H., Certo, M. T., Hubert, C. G., Doyle, L., Getz, M. M., Stoddard, B. L. and Scharenberg, A. M (2009) "High resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display" Nuc. Acids Res. 37 (20): 6871-6880.

For determination of a specificity profile using a selection experiment, see the following reference:

Scalley-Kim, M., McConnell-Smith, A. and Stoddard, B. L. (2007) "Coevolution of homing endonuclease specificity and its host target sequence" J. Mol. Biol. 372 (5): 1305 - 1319.

How should individual values be scaled when entering a PWM into the database?

For data that is derived from relative cleavability data (determined using either in vitro digests or via yeast surface display and flow cytometry), the 'most cleavable' nucleotide at each position (usually but not always the wild-type base) should be given a value of '1.0', and all nucleotides at that same position (measured under identical conditions) should be given values scaled between 0.0 and 1.0.

For data that is derived from frequency of recovery in target selection experiments, the frequency of all four basepairs at each position in the data should add up to 1.0. Those frequencies can be entered directly, and will be automatically scaled to produce a comparable PWM.

References:

PWMs determined via electrophoretic cleavage assays:

Thyme, S., Takeuchi, R., Jarjour, J., Scharenberg, A., Stoddard, B. L. and Baker, D. (2009) "Exploitation of homing endonuclease binding energy for catalysis and design" Nature 461: 1300 - 1304.

PWMs determined via yeast surface display cleavage assays:

Jarjour, J., West-Foyle, H., Certo, M. T., Hubert, C. G., Doyle, L., Getz, M. M., Stoddard, B. L. and Scharenberg, A. M (2009) "High resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display" Nuc. Acids Res. 37 (20): 6871-6880.