I am fusing a protein with a Dam enzyme (http://en.wikipedia.org/wiki/Dam_(methylase)). The idea is that when the protein binds to the DNA, the Dam enzyme will start methylating nearby GATC sites, thus helping identify the protein binding region (using DpnI later on, and microarray technology). However, I have no idea, in theory, how many (bps) will the enzyme traverse across the genome from its starting location. I.e., how far (in terms of bp) from its starting location will the enzyme methylate the GATC sites.

There are methods for finding protein binding regions along the genome that use such technology, such example here: http://www.nature.com/nbt/journal/v18/n4/abs/nbt0400_424.html

  • 1
    $\begingroup$ I am not sure about this but I believe there is not minimum or maximum distance. Each Dam protein will cut and then disassociate from the DNA. I don't think it moves along while remaining connected. If this is correct (again, I am not sure, not my field) then there will be no min/max distance between restriction sites. It will just float around until it finds another DNA molecule and cut the first site it finds. This could be adjacent to the previous one or kilobases away. $\endgroup$ – terdon Nov 14 '12 at 17:26
  • $\begingroup$ get it, thanks terdon. I think you may be right, in my case completely not my field I am a EE... $\endgroup$ – Dnaiel Nov 14 '12 at 17:44
  • 3
    $\begingroup$ Is there a restriction enzyme called Dam? It isn't included in the rather extensive list on Wikipedia. The only Dam that I know of is the Dam methylase of E coli which modifies the A at GATC sites immediately after replication. $\endgroup$ – Alan Boyd Nov 14 '12 at 17:46
  • $\begingroup$ @Alan Boyd - my apologies, I misread your question about Dam, thinking you were referring to DpnI (which is indeed a restriction enzyme with preferential cleavage of methylated DNA). My apologies. $\endgroup$ – user560 Nov 14 '12 at 22:31
  • 1
    $\begingroup$ I'll second Alan's comment, please clarify which enzyme you mean exactly. Dam is a methylase, it doesn't cleave anything. $\endgroup$ – Mad Scientist Nov 14 '12 at 22:57

Dam methyltransferase methylates large DNA molecules in a processive manner, with an effective range of up to 7 kb on either side of an initial methylation reaction.

My answer is taken from this paper:

Urig, S. et al. (2002) The Escherichia coli Dam DNA Methyltransferase Modifies DNA in a Highly Processive Reaction J Mol Biol 319 1085-1096

The authors report on a kinetic analysis of E. coli dam methylase. For anyone using this system as described by the OP, this paper is essential reading. Their conclusion is that the methylation process is highly processive, which is to say that a single dam-DNA interaction leads to multiple methylations before the enzyme dissociates from the DNA.

I'll restrict myself to describing just two of their experiments:

Figure 5 In this experiment they analysed the time course of methylation of an end-labelled 879-mer with 4 GATC sites, using DpnI digestion to detect methylation. In this experiment all DNA was found to be either fully methylated or unmethylated - no evidence for the presence of partially methylated DNA molecules was detected.

Figure 6 In this experiment they analysed the methylation of λ DNA (48.5 kb) and were able to detect partially methylated molecules. A kinetic analysis of the data results in the following conclusions (MTase; methyltransferase):

From these simulations, we [determine] a processivity of nav=3000. This means that after binding to the λ-DNA and starting the one-dimensional diffusion process, one MTase on average meets 3000 dam sites before it dissociates from the DNA. Since the enzyme performs a random walk, it will hit many sites more often than only once and the effective range of processivity corresponds to the square root of nav. Therefore, the dam MTase is able to methylate approximately 55 GATC sites on λ-DNA in a processive reaction in vitro. In this simulation, 4.6 associations of an MTase molecule to the same molecule of λ-DNA are required to obtain fully methylated DNA.

In a random DNA sequence GATC should occur on average every 44 = 256 residues. This suggests an approximate range of (55/2)*256 = 7 kb on each side of an initial methylation reaction.

  • $\begingroup$ couldn't ask for a better answer! thanks so much. $\endgroup$ – Dnaiel Nov 15 '12 at 16:53

After re-reading your question, I suspect you are conflating DNA methylation and restriction digestion. For a more detailed overview of how these two relate to each other, please read the summary page at New England Biolab's website.

The crystal structures of common restriction enzymes do not suggest that they scan along the double-stranded DNA looking for a recognition site. Their reaction kinetics can be thought of as being a more random interaction, whereupon the enzyme comes into contact with a recognition site, then cleaves the DNA is and free to float around until the next reaction.

DNA methylation comes in three common flavours: Dam (DNA adenine methylation), Dcm (DNA cytosine methylation) and CpG methylation. The addition of any of these methyl groups changes can interfere with enzyme recognition of the site. Many enzymes are partially or completely blocked by at least one kind of methylation, while other are completely uninhibited. This is due to the shape of the enzyme and of the DNA.

Now here is where I think you are conflating the two. REbase (Restriction Enzyme database) is indeed the authoritative list on restriction enzyme. You mention Dam cuts at GATC. The sequence GATC is the canonical adenine methylation (Dam) site. As you pointed out, no known restriction enzyme contains Dam. If we instead look up the recognition site, GATC will match with DpnI, a restriction enzyme that produced blunt ends. I think it is DpnI that you are speaking of, which preferentially cleaves Dam methylated DNA (hence why the recognition site GATC also matches Dam mythyltransferases). If the absense of methylated DNA (or if non-methylated DNA is more abundant), DpnI will also digest at GATC sites.

  • $\begingroup$ you are completely right, sorry for the confusion. as a follow up, do you have any idea how far (in terms of bp) dam would move along the genome? thanks! $\endgroup$ – Dnaiel Nov 15 '12 at 0:50
  • $\begingroup$ I rephrased the question based on your perfect suggestion! thanks so much! $\endgroup$ – Dnaiel Nov 15 '12 at 0:58
  • $\begingroup$ It's still unclear to me whether Dam does in fact translate along DNA, or if it too acts like a restriction enzyme, in which case it's more like a probabilistic collision event. $\endgroup$ – user560 Nov 15 '12 at 1:52

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.