Hi,

the modification status of phage DNA is dependent on host modification ability. So if you know, where does your DNA come from (bacteria? Which strain specifically?)

BTW the easiest way is to select restriction enzyme which is not affected by methylation status ;) (which is actually the majority)

1. do you mean in silico or experimentally? For in silico analysis, you just need to know the source of your DNA. For experimental confirmation, you'd need some specialize dsequencing method.

2. it would be good to know, who is your RE supplier, because each of them has different tools and offers slightly different set of enzymes, so it's better to work directly with those you are able to obtain.

For example, NEB has this table, where you can see, which enzymes are sensitive to methylation:

https://www.neb.com/en/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation

3. I think that e.g. SnapGene should take that into account. You can set up the source of your DNA, which affects the methylation status and thus you get information, that particular restriction site would be blocked.

Hi Tomas,

Thank you for the detailed information and resources!

1. I’m working with DNA from the YuA phage, a Pseudomonas phage, with the goal of fragmenting its genomic DNA into 10 or fewer distinct bands of varying lengths. Based on the literature, the YuA phage DNA is modified and resistant to most restriction enzymes. It’s only susceptible to SmaI and Sau3AI, but these produce 93 and 317 fragments, respectively, which result in a smear rather than distinct bands, making them less suitable for my gel assays.

2. We have enzymes from various suppliers, with the majority coming from Thermo Fisher. I will check the tools they offer to identify enzymes that could produce fewer, well-separated bands.

3. I primarily use Benchling, but unfortunately, it doesn’t offer an option for DNA methylation status. I also have access to the free SnapGene Viewer, which I understand has some limitations. If you have any tips on how to handle DNA methylation status or enhance restriction analysis with these tools, it would be very helpful.

4. I checked the NEB table and am thinking of digesting it with an enzyme which is not sensitive to methylation. I am thinking of starting with 500ng of DNA to run the digest as I tried (250ng of Phage DNA) once with SmaI but couldn't see anything clearly on the gel. So, I assume it is not enough.

Any additional suggestions or guidance you can provide would be immensely helpful. Thank you once again for your support!

If you already know that your phage will not be cleaved by most restriction enzymes, then you are likely not dealing with host modification systems or normal dam/dcm like host methylation systems. It could be that the phage is encoding its own methylation or modification system. But that would likely only impact some fraction of restriction enzymes. Sau3A is know to digest methylated DNA. But you could for example look at enzymes that recognize only GC sequences or only AT sequences (or more to the point inhibited by GC methylation or inhibited by AT methylation. Unless you have multiple systems, one of the two sets should work.

However you may be facing something quite different, phage T4 for example substitutes HMC (hydroxymethyl cytosine) for its Cytosine residues and T4 DNA is resistant to cleavage by nearly all restriction enzymes, like your case.

In terms of how much DNA you need, as a rough estimate you should have at least 10-20 ng (or more) PER BAND. So if you are cutting into 4-5 bands then 100ng is enough. If you are cutting into 50 bands, then not nearly enough.

Hi Samiya Siddiqui ,

as Michael J. Benedik suggested, if your DNA is resistant to almost all REs, it is probably something else than the common methylations. In that case you may determine, what is the actual modification. For that you could try to contact Prof. Henrik Nielsen or Prof Peter Brodersen from the University of Copenhagen. They work with plant RNA, but they could be able to help or at least direct you in some better direction.

However, I don't think this will really help you to solve your problem, so the only option I see is to try variety of enzymes and see which ones cut and which ones do not cut your DNA. Try to get as many REs as possible, try contacting colleagues from your Institute or the manufacturers, they could be willing to provide small aliquotes as you need to perform just a couple of reactions with each enzyme. And then just trial and error to see, which REs are able to cut your DNA. That could also inform you about the possible modification.

Alternatively, you could try partial digest with SmaI - digestion for short period of time so that it will not have time to digest your DNA completely. But then you will not have uniform pattern.

Are you sure you need to do restriction digests? Since you have the sequence maybe you can do what you need by PCR?

Are you sure you need to do restriction digests? Since you have the sequence maybe you can do what you need by PCR?

Hi Michael J. Benedik and Tomáš Hluska,

Thanks for the suggestions!

I will try to contact Prof. Henrik Nielsen or Prof Peter Brodersen from the University of Copenhagen. I’m in the process of characterizing a DNA-binding protein from the YuA phage.

I see what you mean, and I agree that testing multiple enzymes could help determine which ones will reliably cut the DNA. However, one challenge I’m facing is that I’d need a significant amount of DNA for these trial-and-error digests, as each one requires 250-500 ng. Since isolating DNA from the phage is time-consuming and yields are relatively low with standard kits, obtaining enough DNA for multiple digests would be quite labor-intensive.

The primary goal of this project is to characterize a seemingly DNA-binding protein from the YuA phage by assessing its specificity for phage-derived DNA sequences. So far, I’ve done an EMSA with commercial PhiX174 DNA in both its single-stranded and double-stranded forms, and now I want to assess its specificity using the phage’s own genomic DNA. To do this, I plan to fragment the YuA genome into distinct bands and run EMSA on these fragments. By observing which bands shift, I’ll be able to pinpoint specific binding regions, which will help further confirm the DNA-binding ability of this protein.

I’m also planning a ChIP-seq experiment to get a clear view of the protein’s binding across the genome. However, I want to employ multiple methods to confirm its binding specificity, so I need at least one additional approach. If anyone has suggestions for software tools or alternative techniques that could help me achieve well-separated DNA fragments or provide insights into this protein’s binding profile, I'd be eager to explore them.

For all the previous suggestions, I am very grateful, and I will definitely keep them in mind as I move forward with this work.

Regards,

Samiya.

Hello Michael J. Benedik and Tomáš Hluska,

I’m a little curious and just wanted to check if you had any thoughts on my approach from the previous response. I don’t mean to bother, but your input would be greatly appreciated.

Thank you!

Sorry I had not seen your previous response. Those details are important to know. My thoughts are to try and pinpoint if there is site specificity in the binding of your protein and then try to map whether that site exists on the phage genome. Did you observe any binding to PhiX174? You could also buy some commercial lambda DNA and try that. But I would probably go right to the ChiP seq. If you can identify some binding sites then you can analyze your phage genome as to whether they exist on the phage and then generate fragments by PCR across the regions you wish to test.

Do keep in mind that the protein may be phage specific, or it may be specifically NOT for its own phage. It also may or may not be impacted whatever modification is happening to your phage DNA. So unfortunately there are lots of options until you can begin narrowing it down.

Can you create a knockout of the gene in your phage and see if there is a phenotype?

No problem at all! Yes, determining the specificity of my protein's binding is definitely a key focus right now, and mapping whether that binding site exists on the phage genome will be crucial for the next steps.

Yes, the protein does seem to bind both the ss and ds DNA of PhiX174, as indicated by a clear band shift in the EMSA. When I looked more closely by adjusting the molar amounts of the protein, I observed that the protein shows specificity toward dsDNA. The shift occurred at lower molar amounts with dsDNA compared to ssDNA, suggesting stronger binding affinity for the double-stranded form.

There are several challenges I’m dealing with at the moment. First, I have to keep purifying my protein frequently because it seems to lose stability even at -80°C, as suggested by the changes in the shift patterns. Unfortunately, I don’t have enough tests yet to determine the activity or even the loss of activity and there’s still much that remains unknown. Additionally, I still need to optimize both the DNA and protein amounts (appropriate molar amounts or ratio) for my EMSAs, which is proving to be quite tricky. So, there’s a lot of work ahead to establish a fully optimized interaction protocol.

The phage deletion mutant has already been made and tested in the lab. There was only a slight delay in phage infection by about 1 hour, but no other significant phenotypic changes were observed. Based on that, I don’t think the protein is essential for the phage, though it might still play a role in host takeover or interactions with the host. What it does and how it does it are questions I still need to answer, but the process is proving to be quite time-consuming and involves a lot of trial and error. I will still try my best to narrow it down with time.

Did you digest the PhiX DNA and shown the binding is just to one band or is it just binding randomly to ds DNA? If the latter then you can probably use any plasmid as template in case that makes your life easier. And if it is binding tightly enough to show a band shift in EMSA on random DNA then most likely it does not have a specific target sequence.

On the other hand if you show specificity to one restriction fragment on PhiX then I would focus on that first and define the binding site.

Hi Samiya Siddiqui ,

I'm sorry, I also missed your answer.

I'm getting on thiner and thiner ice here, but how about to reverse the assay if you have troubles reliably digesting your DNA?

How about you express your DNA-binding protein with some reliable tag (i.e. not His-tag:), which you can easily bind with an antibody (it may even increase stability of your protein). Now, you would fragment your DNA (either digest or I think sonication is used), bind it with your DNA and pull down your protein. Then you release the DNA and sequence. That should give you answer, where your protein binds and if would you clone those DNA fragments into "random" plasmid, you may use them to check, whether there is some modification required for the binding.

Hi Michael J. Benedik

I did not digest the PhiX174 DNA in my initial EMSA experiments.

Based on your suggestion, I will test whether the protein binds randomly to dsDNA by performing EMSA using pUC18 or pUC19 plasmid DNA. This should simplify the process and help clarify the binding behavior.

I really appreciate your idea—it will be very helpful in determining if the protein has specific binding preferences.

I’ll proceed with this approach and let you know what I find.

Thank you again for your insightful suggestions.

Best regards,

Samiya

Hi Tomáš Hluska

Thank you for your thoughtful suggestion!

I think reversing the assay is a great idea. However, I was wondering if there is a specific reason why the His-tag wouldn't work for this approach?

I already have an antibody against the His-tag which I use to perform western blot to confirm my protein purification, so could I still use it for the pull-down? Are there any limitations I should be aware of?

I’ll consider this for my project. Thanks again for the helpful direction!

Best regards, Samiya

I think random DNA is random DNA. You might want to use a template that you can digest into a few fragments (say 2-4) and see if binding is comparable on all fragments. Then you can be comfortable saying it is just a DNA binding protein without specificity. If so, then there is no reason to try and chase down a binding site in your phage.

The reverse experiment proposed by Tomáš Hluska is the right one to try but only if you know there is a specific binding site. If it is binding to any DNA then there is not much point in doing it.

Hi Samiya Siddiqui ,

my only objection is that it may not bind strongly enough and that it may decrease your yield of the protein. But of you have already experience with this combination of protein and tag, then go for it.