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

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<p><a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Description"><font color="blue">Description</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Objective"><font color="blue">Objective</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Explanation"><font color="blue">Explanation</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Bases"><font color="blue">Bases</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Conclusion"><font color="blue">Conclusion</font></a><br><br><b>Synthetic Rally</b><br>
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<p align="justify"><b><font color="black" size="5px">DNA Specific Deletion </font></b>
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<br><a name="Description"></a><b>Description</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
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<p align="justify">One of our activities for human practices consisted in creating a rally that would teach synthetic biology to students from 9th grade. We wanted to show something that was, in some cases, difficult to explain. The plan was to teach them some basic things about molecular biology that would help them understand each of the bases of the rally, including genes and biobricks, and how they could use these to design a circuit for a project. We decided that each of the bases would represent different parts of this year’s circuit, explaining how each of the parts work. For example, base one represented the Promoter; base two, the Riboswitch, and so on. </p>
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<p><br><a name="Objective"></a><b>Objective</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
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<p align="justify">The objective was mainly to help students understand that genetically modified machines work, basically, like a normal machine. We wanted to explain each of the parts that formed our circuit, how each of them worked according to its function, and how every part is necessary for the circuit to work. But all of these are hard to understand when you can’t see the relation between both types of machines. When you tell people who know nothing about genetic engineering that you are building a genetic machine, they have no idea of what to imagine; of how that could work. So we tried to show it to the students in the simplest way. </p>
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<p><br><a name="Explanation"></a><b>Explanation</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
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<p align="justify">The first thing we did when arriving to the school, was to organize all of the materials needed for each one of the bases. While we were doing this, three of our team members went to one of the classrooms that the school had lent us, and explained some basic things about DNA and genes to the students. They explained it in a simple manner, just so the students could understand that these were needed to form the biobricks, and these last ones to form a circuit. But that part was explained later on.</p>
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<p align="justify">After finishing the presentation, the 40 students from the first classroom went outside, to the first base. We divided them in two groups, and gave the first twenty of them small ribands of different colors, to divide them in teams for them to compete. The other twenty were kept waiting, while some of us asked them questions for a survey about transgenic food.</p>
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<p align="justify">We handed out one small card with the drawing of our circuit to each one of the teams from the first group; a card that we would mark, whenever they finished one of the bases, with the points they had earned. The one who came out first place would receive 4 points; second place would receive 3, and third and fourth place would receive 2. We had one person in charge of writing down the points in each card to keep track of who was winning. After explaining all of this, the rally began. </p>
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<p align="justify">The first thing we did at each one of the bases was to identify the name; say if it was the promoter, the terminator, the RBS… and we then gave an explanation of what it was without explaining completely how it worked. At the end of the activity, we would state the relationship between what they had played and the way the actual part of the circuit functioned. Then the one in charged would yell, and they would change bases. When the first group passed to the second base, the group that was kept waiting entered the first base. </p>
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<p><br><a name="Bases"></a><b>Bases</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
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<p align="justify">''BASE 1: PROMOTER'' - The activity was called Streets and Avenues, but the game was changed a little bit. All the teams would be aligned, forming a square, and one person from each team would be standing at each corner. That person would have to find his/her match; someone with the same color inside the square. For example, maybe someone form team Blue, inside the square, would be Yellow. The Yellow person outside would have to find that Blue person before the others found their match, with the path changing just as it does in the common “Streets and Avenues“ game. </p>
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<p align="justify">''How does this relate to the promoter?'' The promoter needs to find the one thing that starts it, for the circuit to start too.  </p>
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<p align="justify">''BASE 2: RIBOSWITCH'' - This activity was called Dragons. Each team had to form a line and hold hands, being really careful not to let go. The person in the very front would be the head, and the last one, with a bandana hanging on their clothes, would be the tail. The objective was that the head would have to take the other team’s bandana, being careful that they didn’t lose theirs. The moment the team lost theirs, they would lose and have to stop right where they were. </p>
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<p align="justify">''How does this relate to the Riboswitch?'' The riboswitch turns the circuit on and off, depending on several conditions. In this game, the team was “On“ while they still had their bandana; they were turned “Off“ when they lost it. </p>
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<p align="justify">''BASE 3: PROTEIN CODING SEQUENCES'' - This base was one of the hardest to fulfill. We designed a “labyrinth“ of conditions that each team would have to pass through. They would have to dress up with objects in some boxes, and depending on what they wore, they would move through the squares. The one who found the perfect combination would win.
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</p>
</p>
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<p align="justify">''How does this relate to the protein coding sequence?'' Protein coding sequences, as stated in the Registry of Standard Biological Parts, encode the amino acid sequence of one particular protein. In the game there was only one perfect combination, one combination that would make you win; and you had to find it. </p>
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<br>
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<p align="justify">“BASE 4: RBS“ - This activity was somewhat simple, yet fun anyway. We would have two people holding a piece of fabric next to two other people, also holding a piece of fabric, and they would be passing along a small ball, in several different ways, to a small box on the other side of the field. For example, the first time it would be walking and passing the ball however they could. The second time, they would have to throw it as higher as possible, and running. The third time would be running backwards, and the fourth would be jumping on one foot. Only one team got to the fifth time, and they had to do it backwards and jumping on one foot. At the end, the team with the greatest amount of balls inside their box, won.</p>
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<p>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.
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<p align="justify">''How does this relate to the RBS?'' The RBS is the place where the ribosomes bind and start the process of translation. The small balls represent the ribosome trying to get to the RBS, the box. </p>
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<p/>
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<p align="justify">“BASE 5: REPORTER“ - The activity was quite simple, and at the same time complicated. We had to build 4 boxes with wires, batteries and a light bulb; only two wires would be connected to the batteries and could turn on the light bulb. There were 20 wires, and the first team who found the combination and turn on the light, would win.</p>
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<p align="justify">''How does this relate to the Reporter?'' The light would turn on whenever the right wires were connected; it acted like a signal. That’s what the reporter does; when the condition is fulfilled, it sends a signal to let you know, just like the light.</p>
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<p align="justify"><b><font color="black" size="5px"> Endonucleases </font></b></p>
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<p align="justify">“BASE 6: TERMINATOR“ - The last activity was just like a game named Doctor, except that we used balloons instead. Each team would have to get in a closed circle, but instead of holding hands directly, they would hold a large balloon. Then they would have to tangle, without letting go of the balloons, as much as possible. The “doctor“ would then have to undo their “knot“, without breaking the circle. The first ones to blow up or let go of their balloon would lose. </p>
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<p align="justify">''How does this relate to the terminator?“ The terminator causes transcription to stop; it sort of “breaks it off“. In the game, “transcription“ stopped whenever the person blow up or let go of the balloon. </p>
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<center>
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<p><br><a name="Conclusion"></a><b>Conclusion</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
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<img src= "https://static.igem.org/mediawiki/2014/d/d9/Tablauanl212114.png"/>
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<p align="justify">At the end of the rally, we gave an explanation of the whole circuit; how every part was connected to make it function. We answered questions, we took a picture, and we prized the winner team with a box of cookies. Some of them asked if we had a page on Facebook, and others just thanked us for coming. </p>
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</center>
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<p align="justify">One of the girls who was helping us with the rally, was not from the team. She was from second semester, and was really interested in iGEM when she found out about it, so she asked if she could come and help. She was of great support; taking pictures and helping us organize everything. At the end it all worked out, even though the day before we were all going crazy because most of the things were missing. It was a really fun experience. </p>
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<p> 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).  
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</p>
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<p align="justify">
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According to their characteristics, they are divided into four types, from I to IV.
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Moreover, each of them has their own specific applications. The most studied and
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used are type II restriction enzymes, because they recognize a specific palindromic
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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).
 +
</p>
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<p align="justify">
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Among type 2 restriction enzymes, there are those who can cut just one strand,
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which is called nicking. They are generally named with an N prefix, for example,
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N.bstSEI (13).
 +
</p>
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<p align="justify">
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Restriction enzymes can recognize symmetric and asymmetric sequences
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(10). One way of classifying those that recognize asymmetric
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sequences is in 5 classes according to their characteristics,  
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which are shown in the table.
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</p>
 +
 
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<center>
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<img src= "https://static.igem.org/mediawiki/2014/d/d1/Tablaissacuanl14.png"/>
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</center>
 +
 
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<p align="justify">
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Type II restriction enzymes have come to be used so much in the molecular
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biology field, that commercially they are the most exploited. But even among them,
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there can be advantages and disadvantages. For example, if an enzyme has a  
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recognition site of a few nucleotides, it is better suited for diagnostic trials than for
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genetic engineering. For this reason the use and research of new enzymes has
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begun, along with the de novo design of others.
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</p>
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<p align="justify">
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The mode of action of type II restrictions enzymes can be imitated with artificially
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designed enzymes. For example, the union of proteins and/or peptides that
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recognize certain sequences with others that have the capacity of excising DNA.  
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</p>
 +
 
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<p align="justify">
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In our project we plan to use endonucleases, together with polypeptides that
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recognize longer sequences than restriction enzymes, in order to increase the
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specificity.
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</p>
 +
 
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<p align="justify"><b><font color="black" size="5px"> Zinc- Finger Nucleases (ZFN)  </font></b></p>
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<p align="justify">
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Zinc-finger nucleases are agents that have been used for DNA modification by
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means of the fusion of a zinc finger, designed or preexistent, with the active
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domain of the Fokl enzyme; this Phusion molecule is called “Zinc Finger Nuclease”
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or ZFN (4). They have been used in different organisms, from
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animals to plants (8) with the purpose of modifying them, for
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example, through an integration of complete genes (9).  
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</p>
 +
 
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<p align="justify">
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An important aspect to be considered before using this technology, is that in order
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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
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interact in an intermediate site. There are also other advantages, like its high
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specificity due to the ability to design binding sites of over 18 bp (17)
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and because they normally only cut once they have bound to the specific site
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(18). On the other hand, when the ZFP has not joined its specific
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site, Fokl remains as a monomer even to the 15 µM (3),  
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making the appearance of cuts in non-specific sites more difficult.  
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</p>
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<p align="justify">
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However, it is necessary to mention that some possible disadvantages exist, ones
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that may surface in spite of good planning. One of them is that the effectiveness of
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a ZFN in one species doesn’t it will function in others (--). Another downside may
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be that the designed ZFNs work, but once they homodimerize they cut sites they
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weren’t designed for, becoming toxic for the cell (1). Due to this,
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the designing of new ZFN requires experimentation to assure its correct
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functioning.
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</p>
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<p align="justify">
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In our project ZFNs will be used, and to avoid the problems mentioned above, we
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will use ZFNs that have been previously tested in E. coli. This is because our aim
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is to prove the utility of said protein in our project.  
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</p>
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<p align="justify"><b><font color="black" size="5px"> TALEN (Transcription Activator-Like Effector Nucleases): </font></b></p>
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<center>
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<img src= "https://static.igem.org/mediawiki/2014/2/2e/Captura_de_pantalla_2014-10-17_a_la%28s%29_20.55.52.png"/>
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</center>
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<p align="justify">
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There are a number of ways to generate ruptures in double stranded
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DNA, one of them is the technology known as TALEN. They are phusion
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proteins composed of a DNA binding domain called TALE (Transcription
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activator-like effector), that is naturally found in the pathogenic plant
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bacteria Xanthomonas, and the Fok l nuclease domain (1)(2)(3).  
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</p>
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<p align="justify">
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The TALE domain can be modified in order to bind to a specific site in
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the DNA strand. This modification is based in the change of two amino
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acids in the RVD or repetitive variable Di-residue region (1) (2) inside of
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each TALEN. Two amino acids have been determined to correspond to
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each of the 4 nitrogen bases that make up the DNA strand, this means
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the TALE domain can be directed to practically any sequence (2)(3).
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Each TALE domain consists of approximately 33-35 repeated amino
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acids in the RVD region (1), so each TALEN is able to recognize a site
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specific region of up to 35 base pairs, which highlights its high
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specificity. If a single TALEN were directed to a DNA strand, this would  
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just cause a nick that would in no way affect the organism, making our
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whole experimental goal void. In order to solve this, 2 TALEN are going
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to be directed to both strands in opposing sites (Figure 1), separated by
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a 5 base pair spacer, to rupture the DNA molecule completely. The Talen
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technology is classified in the new era genome edition, along with the
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Zinc Finger Nucleases (ZFN) and the CRISP/Cas system (1).  
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</p>
 +
 
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<p align="justify">
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The two criteria that led us to select this tool as the primary factor in
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our project were the high specificity of the TALE domain, and the lack of
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literature reporting the use of TALEN technology to generate double
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ruptures in a bacterial model.
 +
</p>
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<p align="justify"><b><font color="black" size="5px"> Intracellular degradation of lineal DNA </font></b></p>
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<p align="justify">
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When endonucleases break double stranded DNA in the middle of its sequence,  
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lineal DNA fragments remain, which are degraded by their previously mentioned
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counterparts, the exonucleases, which are enzymes with nuclease activity that
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fragment DNA by their ends.  
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</p>
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<p align="justify">
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In vivo, this mechanism is used to fragment free DNA remaining after phenomena
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like recombination, which consists in the excision of a sequence from the  
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chromosome when it is damaged and its substitution with a newly synthesized
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chain.  
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</p>
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<p align="justify">
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However, the exonuclease system is also used for other purposes in the cell, like
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the degradation of exogeneous DNA, the destruction of DNA after the digestion of
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bacteriophages, virus, lineal plasmids, or RNA degradation. Therefore, the
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presence of exonucleases, its activity, quantity and class will determine the amount
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of time lineal DNA will last and thus, the velocity in which it will be inactivated.  
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</p>
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<p align="justify">
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The role of these exonucleases in the project is essential, because even if the  
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endonucleases can fragment DNA, the coding DNA will remain and the
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reprogramation scheme will fail.  
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</p>
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<p align="justify">
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In bacterias, this mechanism is well studied, because the group of rec proteins is in
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charge of the exnonuclease activity, associated in Exo complexes. In particular, the  
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ExoV nuclease, which is the most powerful known exonuclease, with helicase
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activity and single stranded DNA endonuclease activity, is capable of degrading up  
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to 1kb per second and of separating up to 30 kbp per union event. So, even if the  
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ATP and calcium concentrations aren´t very high, the system will ensure that lineal
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DNA will be quickly degraded.  
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</p>
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<p align="justify">
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On the other hand, we have expression systems or transgenic organisms, like
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yeasts or cultures (especially cultures). And having repairing DNA systems, they
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also require exonucleases.
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</p>
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<p align="justify">
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<i>PLANTS</i>
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<br>
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Plants in general lack a variety of double stranded DNA exonucleases, but they
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have different helicases (at least 5 homologous of RecQ) and single stranded DNA
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endonucleases.
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</p>
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<p align="justify">
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However, a similar protein to hWRN has been described in Arabidopsis thaliana,
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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
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endonucleases capable of generating sticky ends must be made to make this DNA
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degradation in plants successful. Also, if further investigation concludes that plants
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lack an efficient double stranded DNA degradation system, it must be included in
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the RSP system.
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</p>
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<p align="justify">
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Other nucleases in plants exist, but they are not well studied, or they´re not present
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in the whole organism.
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</p>
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<p align="justify">
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<i>YEAST AND OTHER EUKARYOTES</i>  
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<br>
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In yeasts and other eukaryotes, the Exol protein functions as part of the
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recombining system, but in plants and bacteria, this protein has a single and
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double stranded desoxyribonuclease activity from 5’ to 3’, as well as a
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desoxyribonuclease activity from 3’ to 5’. Also, several single stranded
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exonucleases as well as endogenous endonucleases have been described.
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</p>
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<p align="justify">
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From these information, we can infer that the endogenous DNA degradation
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systems, of the organisms that are going to be used, are capable of digesting the  
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single stranded DNA that remains after the endonuclease digestion of the system.  
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And even if this is not the case, known exonucleases can be added to the RSP
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systems to make this degradation possible.  
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</p>
 +
 
 +
<p align="justify"><b><font color="black" size="5px"> Bibliography </font></b></p>
 +
 
 +
<ol>
 +
<li>
 +
[1] Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J.K. & Carroll, D (2006)
 +
Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics, 172, 2391-2403.
 +
</li>
 +
 
 +
<li>
 +
[2] Bitinaite J, Wah DA, Aggarwal, AK, Schildkraut I (1998) FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci., 95: 10570-10575.
 +
</li>
 +
 
 +
<li>
 +
[3] Kaczorowski, T, Skowron, P & Podhajska, AJ (1989) Purification and characterization of the FokI restriction endonuclease. Gene, 80: 209-216.
 +
</li>
 +
 
 +
<li>
 +
[4] Kim YG, Cha J, Chandrasegaran S. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. 93: 1156-1160.
 +
</li>
 +
 
 +
<li>
 +
[5] Kuzminov A, Stahl FW. (1997) “Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation”. Journal of Bacteriology 179(3): 880-888
 +
</li>
 +
 
 +
<li>
 +
[6] Li X, Hejna J, Moses RE. (2005) “The yeast Snm1 protein is a DNA 5’-exonuclease”. DNA Repair, 4: 163-170
 +
</li>
 +
 
 +
<li>
 +
[7] Matsushima R, Tang LY, Zhang Lingang, Yamada H, Twell D, Sakamoto W.
 +
(2011) “A conserved Mg2+
 +
-dependent exonuclease degrades organelle DNA
 +
during Arabidopsis pollen development” . The Plant Cell 23:1608-1624.
 +
</li>
 +
 
 +
<li>
 +
[8] Miller JC, Holmer MC, Wang J, Guschin DY, Lee YL, Rupniewski I,
 +
Beasejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar
 +
EJ (2007) An improved zinc fingernuclease architecture for highly specific
 +
genome editing. Nature Biotechnology, 25(7): 778 785.
 +
</li>
 +
 
 +
<li>
 +
[9] Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Grgory PD, Urnov
 +
FD, Holmes MC (2007) Targeted gene addition into a specified location in
 +
the human genome using designed zinc finger nucleases. Proc. Natl. Acad.
 +
Sci. 104: 3055-3060.
 +
</li>
 +
 
 +
<li>
 +
[10] Pingoud A, and A. Jeltsch (2001) Structure and function of type II restriction
 +
endonucleases. Nucleic Acid Research, 29(18): 3705-3727.
 +
</li>
 +
 
 +
<li>
 +
[11]Plchova H, Hartung F, Puchta H (2003) “Biochemical characterization of an
 +
exonuclease from Arabidopsis thaliana reveals similarities to the DNA
 +
exonuclease of the human Werner syndrome protein”. The Journal of
 +
Biological Chemistry 278(45): 44128-44138
 +
</li>
 +
 
 +
<li>
 +
[12]Qiu J, Qian Y, Chen V, Guan MX, Shen B. (1999) “Human exonuclease 1
 +
functially complements its yeast homologues in DNA recombination, RNA
 +
primer removal and mutation avoidance”. The Journal of Biological
 +
Chemistry, 274(25):17893-17900
 +
</li>
 +
 
 +
<li>
 +
[13]Roberts RJ, M Belfort, T Bestor, AS Bhagwat , TA Bickle , J Bitinaite, RM
 +
Blumenthal, S Degtyarev , DT Dryden and K Dybvig (2003) A nomenclature for
 +
restriction enzymes, DNA methyltransferases, homing endonucleases and their
 +
genes. Nucleic Acid Research, 21: 1805-1815.
 +
</li>
 +
 
 +
<li>
 +
[14]Roman LJ, Kowalczykowski SC. (1989) “Characterization of the helicase
 +
activity of the Escherichia coli RecBCD enzyme using a novel helicase
 +
assay.” Biochemistry, 28: 2863-2873
 +
</li>
 +
 
 +
<li>
 +
[15]Simmon VF, Lederberg. (1972) “Degradation of bacteriophage lambda
 +
deoxyribonucleic acid after restriction by Escherichia coli K-12”. Journal of
 +
Bacteriology, 112(1): 161-169
 +
</li>
 +
 
 +
<li>
 +
[16]Skarstad K, Boye E. (1993) “Degradation of individual chromosomes in recA
 +
mutants of Escherichia coli. “ Journal of Bacteriology, 175(17): 5505-5509
 +
</li>
 +
 
 +
 
 +
<li>
 +
[17] Urnov F.D., Miller JC, Lee YL, Beansejour CM, Rock JM, Augustus S, Jamieson
 +
AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous
 +
human gene correction using designed zinc-finger nucleases. Nature, 435: 646–
 +
651.
 +
</li>
 +
 
 +
<li>
 +
[18] Vanamee ES, Santagata S, Aggarwal AK (2001) FokI requires two specific DNA
 +
sites for
 +
cleavage. J. Mol. Biol., 309, 69–78.
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</li>
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Latest revision as of 03:24, 18 October 2014

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.

Bibliography

  1. [1] Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J.K. & Carroll, D (2006) Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics, 172, 2391-2403.
  2. [2] Bitinaite J, Wah DA, Aggarwal, AK, Schildkraut I (1998) FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci., 95: 10570-10575.
  3. [3] Kaczorowski, T, Skowron, P & Podhajska, AJ (1989) Purification and characterization of the FokI restriction endonuclease. Gene, 80: 209-216.
  4. [4] Kim YG, Cha J, Chandrasegaran S. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. 93: 1156-1160.
  5. [5] Kuzminov A, Stahl FW. (1997) “Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation”. Journal of Bacteriology 179(3): 880-888
  6. [6] Li X, Hejna J, Moses RE. (2005) “The yeast Snm1 protein is a DNA 5’-exonuclease”. DNA Repair, 4: 163-170
  7. [7] Matsushima R, Tang LY, Zhang Lingang, Yamada H, Twell D, Sakamoto W. (2011) “A conserved Mg2+ -dependent exonuclease degrades organelle DNA during Arabidopsis pollen development” . The Plant Cell 23:1608-1624.
  8. [8] Miller JC, Holmer MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beasejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc fingernuclease architecture for highly specific genome editing. Nature Biotechnology, 25(7): 778 785.
  9. [9] Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Grgory PD, Urnov FD, Holmes MC (2007) Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl. Acad. Sci. 104: 3055-3060.
  10. [10] Pingoud A, and A. Jeltsch (2001) Structure and function of type II restriction endonucleases. Nucleic Acid Research, 29(18): 3705-3727.
  11. [11]Plchova H, Hartung F, Puchta H (2003) “Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein”. The Journal of Biological Chemistry 278(45): 44128-44138
  12. [12]Qiu J, Qian Y, Chen V, Guan MX, Shen B. (1999) “Human exonuclease 1 functially complements its yeast homologues in DNA recombination, RNA primer removal and mutation avoidance”. The Journal of Biological Chemistry, 274(25):17893-17900
  13. [13]Roberts RJ, M Belfort, T Bestor, AS Bhagwat , TA Bickle , J Bitinaite, RM Blumenthal, S Degtyarev , DT Dryden and K Dybvig (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acid Research, 21: 1805-1815.
  14. [14]Roman LJ, Kowalczykowski SC. (1989) “Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay.” Biochemistry, 28: 2863-2873
  15. [15]Simmon VF, Lederberg. (1972) “Degradation of bacteriophage lambda deoxyribonucleic acid after restriction by Escherichia coli K-12”. Journal of Bacteriology, 112(1): 161-169
  16. [16]Skarstad K, Boye E. (1993) “Degradation of individual chromosomes in recA mutants of Escherichia coli. “ Journal of Bacteriology, 175(17): 5505-5509
  17. [17] Urnov F.D., Miller JC, Lee YL, Beansejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 435: 646– 651.
  18. [18] Vanamee ES, Santagata S, Aggarwal AK (2001) FokI requires two specific DNA sites for cleavage. J. Mol. Biol., 309, 69–78.
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