Team:UANL Mty-Mexico/project/DNA-Program-Supression

DNA Specific Deletion

The DNA, just like a lot of other molecules, suffers from deletion; be it to repair, insert a fragment, recombine or as a defense strategy. There are multiple causes for this, but the one that interest us is due to enzymatic action, among which we find exonucleases, restriction enzymes, and other molecules that will be revised shortly.

Endonucleases

Nucleases are enzymes with the ability to fragment DNA through phosphodiester bond ruptures. When the cutting site is in the 5’ or 3' end, it’s called an exonuclease; on the other hand, if it's inside the DNA strand, it´s called an endonuclease. Among endonucleases, restriction enzymes, which can recognize specific DNA sequences, have been of utmost interest in the manipulation of DNA, from polymorphism identification (molecular diagnosis) to the construction of new DNA sequences (genetic engineering).

According to their characteristics, they are divided into four types, from I to IV. Moreover, each of them has their own specific applications. The most studied and used are type II restriction enzymes, because they recognize a specific palindromic sequence and they generally inside this sequence. On the other hand, type I enzymes cut approximately at a 1000 bp distance, while type III cuts at a 24-26 bp distance. Finally, type IV has low specificity and only cuts methylated DNA (13).

Among type 2 restriction enzymes, there are those who can cut just one strand, which is called nicking. They are generally named with an N prefix, for example, N.bstSEI (13).

Restriction enzymes can recognize symmetric and asymmetric sequences (10). One way of classifying those that recognize asymmetric sequences is in 5 classes according to their characteristics, which are shown in the table.

Type II restriction enzymes have come to be used so much in the molecular biology field, that commercially they are the most exploited. But even among them, there can be advantages and disadvantages. For example, if an enzyme has a recognition site of a few nucleotides, it is better suited for diagnostic trials than for genetic engineering. For this reason the use and research of new enzymes has begun, along with the de novo design of others.

The mode of action of type II restrictions enzymes can be imitated with artificially designed enzymes. For example, the union of proteins and/or peptides that recognize certain sequences with others that have the capacity of excising DNA.

In our project we plan to use endonucleases, together with polypeptides that recognize longer sequences than restriction enzymes, in order to increase the specificity.

Zinc- Finger Nucleases (ZFN)

Zinc-finger nucleases are agents that have been used for DNA modification by means of the fusion of a zinc finger, designed or preexistent, with the active domain of the Fokl enzyme; this Phusion molecule is called “Zinc Finger Nuclease” or ZFN (4). They have been used in different organisms, from animals to plants (8) with the purpose of modifying them, for example, through an integration of complete genes (9).

An important aspect to be considered before using this technology, is that in order to cut it needs to dimerize with the functional domain of another Fokl (2). That’s why it´s required to design adjacent ZFNs whose Fokl domains interact in an intermediate site. There are also other advantages, like its high specificity due to the ability to design binding sites of over 18 bp (17) and because they normally only cut once they have bound to the specific site (18). On the other hand, when the ZFP has not joined its specific site, Fokl remains as a monomer even to the 15 µM (3), making the appearance of cuts in non-specific sites more difficult.

However, it is necessary to mention that some possible disadvantages exist, ones that may surface in spite of good planning. One of them is that the effectiveness of a ZFN in one species doesn’t it will function in others (--). Another downside may be that the designed ZFNs work, but once they homodimerize they cut sites they weren’t designed for, becoming toxic for the cell (1). Due to this, the designing of new ZFN requires experimentation to assure its correct functioning.

In our project ZFNs will be used, and to avoid the problems mentioned above, we will use ZFNs that have been previously tested in E. coli. This is because our aim is to prove the utility of said protein in our project.

TALEN (Transcription Activator-Like Effector Nucleases):

There are a number of ways to generate ruptures in double stranded DNA, one of them is the technology known as TALEN. They are phusion proteins composed of a DNA binding domain called TALE (Transcription activator-like effector), that is naturally found in the pathogenic plant bacteria Xanthomonas, and the Fok l nuclease domain (1)(2)(3).

The TALE domain can be modified in order to bind to a specific site in the DNA strand. This modification is based in the change of two amino acids in the RVD or repetitive variable Di-residue region (1) (2) inside of each TALEN. Two amino acids have been determined to correspond to each of the 4 nitrogen bases that make up the DNA strand, this means the TALE domain can be directed to practically any sequence (2)(3). Each TALE domain consists of approximately 33-35 repeated amino acids in the RVD region (1), so each TALEN is able to recognize a site specific region of up to 35 base pairs, which highlights its high specificity. If a single TALEN were directed to a DNA strand, this would just cause a nick that would in no way affect the organism, making our whole experimental goal void. In order to solve this, 2 TALEN are going to be directed to both strands in opposing sites (Figure 1), separated by a 5 base pair spacer, to rupture the DNA molecule completely. The Talen technology is classified in the new era genome edition, along with the Zinc Finger Nucleases (ZFN) and the CRISP/Cas system (1).

The two criteria that led us to select this tool as the primary factor in our project were the high specificity of the TALE domain, and the lack of literature reporting the use of TALEN technology to generate double ruptures in a bacterial model.

Intracellular degradation of lineal DNA

When endonucleases break double stranded DNA in the middle of its sequence, lineal DNA fragments remain, which are degraded by their previously mentioned counterparts, the exonucleases, which are enzymes with nuclease activity that fragment DNA by their ends.

In vivo, this mechanism is used to fragment free DNA remaining after phenomena like recombination, which consists in the excision of a sequence from the chromosome when it is damaged and its substitution with a newly synthesized chain.

However, the exonuclease system is also used for other purposes in the cell, like the degradation of exogeneous DNA, the destruction of DNA after the digestion of bacteriophages, virus, lineal plasmids, or RNA degradation. Therefore, the presence of exonucleases, its activity, quantity and class will determine the amount of time lineal DNA will last and thus, the velocity in which it will be inactivated.

The role of these exonucleases in the project is essential, because even if the endonucleases can fragment DNA, the coding DNA will remain and the reprogramation scheme will fail.

In bacterias, this mechanism is well studied, because the group of rec proteins is in charge of the exnonuclease activity, associated in Exo complexes. In particular, the ExoV nuclease, which is the most powerful known exonuclease, with helicase activity and single stranded DNA endonuclease activity, is capable of degrading up to 1kb per second and of separating up to 30 kbp per union event. So, even if the ATP and calcium concentrations aren´t very high, the system will ensure that lineal DNA will be quickly degraded.

On the other hand, we have expression systems or transgenic organisms, like yeasts or cultures (especially cultures). And having repairing DNA systems, they also require exonucleases.

PLANTS
Plants in general lack a variety of double stranded DNA exonucleases, but they have different helicases (at least 5 homologous of RecQ) and single stranded DNA endonucleases.

However, a similar protein to hWRN has been described in Arabidopsis thaliana, an exonuclease capable of digesting the delayed strand of the double stranded DNA. But it hardly digests single stranded or blunt ended DNA, so specific endonucleases capable of generating sticky ends must be made to make this DNA degradation in plants successful. Also, if further investigation concludes that plants lack an efficient double stranded DNA degradation system, it must be included in the RSP system.

Other nucleases in plants exist, but they are not well studied, or they´re not present in the whole organism.

YEAST AND OTHER EUKARYOTES
In yeasts and other eukaryotes, the Exol protein functions as part of the recombining system, but in plants and bacteria, this protein has a single and double stranded desoxyribonuclease activity from 5’ to 3’, as well as a desoxyribonuclease activity from 3’ to 5’. Also, several single stranded exonucleases as well as endogenous endonucleases have been described.

From these information, we can infer that the endogenous DNA degradation systems, of the organisms that are going to be used, are capable of digesting the single stranded DNA that remains after the endonuclease digestion of the system. And even if this is not the case, known exonucleases can be added to the RSP systems to make this degradation possible.

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