- DNA questions
- Eukaryotic gene transcription: Going from DNA to mRNA
- Molecular structure of DNA
- Antiparallel structure of DNA strands
- Telomeres and single copy DNA vs repetitive DNA
- Leading and lagging strands in DNA replication
- Transcription and mRNA processing
- Speed and precision of DNA replication
- Translation (mRNA to protein)
- Differences in translation between prokaryotes and eukaryotes
- DNA repair 1
- DNA repair 2
- Semi conservative replication
- Protein modifications
- Jacob Monod lac operon
- DNA structure and function
DNA repair 1
Created by Efrat Bruck.
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- Why was an exonuclease used in the mismatch repair mechanism? Since the mutation was in the middle of the strand, shouldn't an endonuclease have been used instead?(91 votes)
- At3:22, the presenter was correct. When DNA polymerase III recognizes that it has made a mistake in replicating the strand, it can act as an EXOnuclease and remove the nucleotide that it just added to the strand. It then inserts the correct nucleotide in its place. Because this happens during synthesis of the strand, the nucleotide that it is removing is at the 3' of the strand being synthesized and can be removed by the (3'-5') exonuclease activity of DNA pol III. So, while DNA polymerase III synthesizes strands in the 5' to 3' direction, it can remove nucleotides in the 3'-5' direction.
At8:48the presenter was also correct, although with some clarification. When an incorrect base is recognized, there are actually two types of mismatch repair that can occur: base excision repair and nucleotide excision repair.
In base excision repair, a protein called a glycosylase recognizes a mismatched base. It then removes the base, and recruits an endonuclease (called an AP endonuclease) to make a small cut on the phosphodiester backbone. An EXOnuclease is then associated, and it removes the bases surrounding the nick. This is key, because the exonuclease is doing the important work here. So, an endonuclease makes a small nick so that there is an "end" for the exonuclease to work off of, and the exonuclease then "chews up" the nicked strand in area radiating away from the nick. Then, DNA polymerase I comes in and fills the "chewed up" area in, and ligase fills in the last phosphodiester bond.
Nucleotide excision repair is a bit more complex, but suffice to say that it involves a protein complex (UvrABC in E. coli) called an exinuclease. It works by cutting the backbone in two places approximately equidistant from the wrong base and removing the section including the incorrectly paired base.
Sources: Hartwell Genetics, 5th Edition.(73 votes)
- If DNA polymerase I has exonuclease activity, how is it able to remove the RNA primers since they aren't at the ends of the strand? I thought it was RNAse H that went back to remove the primers and then have the gap filled by DNA ligase.(16 votes)
- "In the replication process, RNAse H removes the RNA primer (created by Primase) from the lagging strand and then Polymerase I fills in the necessary nucleotides between the Okazaki fragments (see DNA replication) in 5' -> 3' direction, proofreading for mistakes as it goes. It is a template-dependent enzyme - it only adds nucleotides that correctly base pair with an existing DNA strand acting as a template. DNA Ligase then joins the various fragments together into a continuous strand of DNA." -Wikipedia (DNA Pol I page)(2 votes)
- At 47s, she says that helicase is the enzyme that unwinds DNA. I think it would be more accurate to say topoisomerase "unwinds" the DNA. Helicase actually separates the two strands by breaking the hydrogen bonds between them. Is this correct?(6 votes)
- What she says is correct- helicase unwinds the DNA. Unwinding the DNA, and separating the DNA strands mean the same thing.
Topoisomerase will release the supercoils that are formed ahead, due to the unwinding action of the helicase. Topoisomerase cuts the phosphodiester bonds in one or two strands of the DNA, thus releasing the supercoils, and then it seals these nicks back.(9 votes)
- At8:49shouldn't it be endonuclease?(8 votes)
- An endonuclease presents a cut at the dsDNA which gives the DNA exonuclease an "end" to work with.(5 votes)
- What are the major differences between DNA Pol I and DNA Pol III?(3 votes)
- Are we going to have to know specifically how the DNA strand is cut for the new MCAT?(2 votes)
- Good question. My guess is no (at least for the very specific details). Most of the information should be presented in the passage (if tested on this topic). With all the information that the new MCAT will be testing us on, it seems crazy to ask about specifics like these (DNA Polymerase I, II, III, and what each one does, etc.). That stuff should be stated in a passage. Always keep the big picture in mind and know general details.(5 votes)
- If DNA polymerase is capable of chaining together nucleotides during replication, why is DNA ligase needed to complete the sugar bond after mismatch repair mechanism proteins remove the incorrect nucleotide and DNA polymerase adds the correct nucleotide? Wouldn't DNA polymerase be capable of attaching the nucleotide to the backbone?(2 votes)
- DNA polymerase can only add a nucleotide to the 3' end of a nucleotide in mismatch repair mechanism the new nucleotide needs to have the bond rejoined at both the 5' and 3', which requires DNA ligase. Kind of similar to what happens during replication of the lagging strand.(3 votes)
- At1:07, why is there an RNA primer for synthesizing DNA?(2 votes)
- So at the beginning of the the DNA replication process, comes a DNA Primase to create the RNA primers as the beginning of the daughter strand before DNA polymerase III is able to come and synthesize/add DNA nucleotides until the new/daughter strand is formed.
***If you watch any night show (like Jimmy Fallon's for example), Think of Steve Higgins, the one who introduces Jimmy with kind words before Jimmy comes out and starts hosting the show. Now lets relate this to DNA replication.......DNA primase (Steve) comes and lays down RNA primers (the kind words) before DNA pol III (Jimmy) comes and starts synthesizing DNA (starts the show). So without DNA primase (Steve) there wouldn't be RNA primers (kind words) and DNA pol III (jimmy) won't show up and the replication (the show) will never go on.(2 votes)
- Does DNA polymerase III remove just the nucleic acid or the entire nucleotide, phosphate and sugar included?(2 votes)
- At0:45, she says DNA helicase 'unwinds' the double strand. Isn't that supposed to be unzips? shouldn't topoisomerase unwind?(3 votes)
- Let's take a look at a segment of DNA that's in the process of being replicated. I want to focus in particular on the enzyme that replicates DNA. That enzyme is DNA polymerase. Actually there are a few different types of DNA polymerases, and the one that we're looking at right now is DNA polymerase III. DNA polymerase III synthesizes new DNA, and it also has the ability to proofread, or kind of check the DNA it's putting together and make sure there are no mistakes in it. But before we get into that, let's just orient ourselves and quickly summarize the diagram that we're looking at. This enzyme over here is DNA helicase. That's the enzyme that unwinds the double-stranded DNA, so that DNA polymerase can then come in and started replicating. Right over here you can see I drew the backbone in a different color. That's the RNA primer. Let's just label the DNA strand that's being synthesized, it's synthesized from 5 prime to 3 prime. Actually, the bottom strand of DNA is synthesized in the same time as the top strand, but I just left that out of the drawing to keep things simple. Let's say that the yellow bases represent the nitrogen base thymine. Let's say that the orange bases represent cytosine. The green ones represent adenine. And the blue ones represent guanine. Thymine and cytosine are the pyramidines. They are composed of a single ring structure, so they're made up of one ring that has six sides to it. Then adenine and guanine are the purines. They are a double-ringed structure. They're composed of one ring with six sides to it, and then that ring is attached to another ring that has five sides to it. Actually, these structures are a little bit more complex. There are other atoms in it, and there are some double bonds, but we're just going to keep things simple for now and leave it at that. Let's get back to our DNA that's being replicated. Right over here I left a space. I didn't put the nucleotide in. Let's say that by accident, instead of it being paired up with the proper base, which is adenine, it accidentally gets paired up with a guanine. That's a mistake. DNA polymerase III actually has the ability to sense if it made a mistake, and if it does realize that it's going to go backwards. It's going to actually remove the incorrect base and replace it with the correct base. So let's do that. It's going to remove the incorrect base and replace it with the correct one. Of course, remember the nitrogen base is attached to the sugar backbone. This activity that I just described to you is called exonuclease activity. Nuclease, that tells us that means the ability to remove a nucleotide. Exo, just going to underline that. Exo tells us that it can remove a nucleotide, but only from the end of a DNA strand. It was able to remove the nucleotide because it was at the end of a strand. This is in contrast to endonuclease activity. An endonuclease can actually remove a nucleotide from the middle of a DNA strand. So it would be able to remove a nucleotide from right over here, for example. Just keep that in mind because we're going to come across some endonucleases as well. Anyway, back to our exonuclease activity. If we want to be more specific, the exonuclease activity of DNA polymerase III is actually 3 prime to 5 prime exonuclease activity. The reason it's called 3 prime to 5 prime exonuclease is because when DNA polymerase 3 makes that correction it has to move backwards in the 3 prime to 5 prime direction in order to do that. There's another enzyme, DNA polymerase I. I'm just going to abbreviate polymerase with POL. DNA polymerase I also has exonuclease activity. DNA polymerase I is actually the enzyme that will remove the RNA primer at the end of replication. Just as a side fact, the exonuclease activity of DNA polymerase I is actually in the 5 prime to 3 prime direction. If you want, you can just keep that in mind. DNA polymerase III and DNA polymerase I are both able to repair or fix mistakes that happen during DNA replication. Just to give you some perspective as to how often this occurs with and without repairs, normally we'll have a mistake happening in replication between 1 in 100,000 bases to 1 in 1 million bases. That's normally the amount of mistakes that would occur. But, with the repair mechanisms of DNA polymerase III and DNA polymerase I, this is reduced to a mistake that happens once in about 100 million bases. They are very, very effective at lowering the error rate in DNA replication. The next question I want to ask is what if this mistake over here was somehow not corrected during replication? Maybe there was something wrong with one of the enzymes, something happened and that mistake was actually sustained. Let's take a look at that. Here is a piece of DNA with our mistake incorporated into it. Before we discuss if this mistake can be corrected or not, let's see what happens if this mistake is not corrected. Right here we have our original DNA. We're replicating it. Let's just say that this strand over here is the same as that strand. Let's say that the bottom strand in our original DNA is the same as this strand. Let's look first at the newly replicated DNA on the left. We have right over here a thymine base. Assuming DNA was replicated properly, it's going to have an adenine complementary to it. Now let's take a look at the DNA on the right. On the bottom we had a guanine, and it's going to be paired up, hopefully, with the correct base, which is a cytosine. Now, let's just quickly look back at our original DNA. We were supposed to have a thymine with it's complementary adenine, and actually, that's exactly what we got over here. Just going to circle it. So this DNA is actually in the correct sequence. But look at the DNA over here on the right. This is not correct. This is a mutation. This is an example of how mutations can occur if the DNA repair mechanisms are not working properly. Let's go back to our original question. Can we fix the original mistake so that this mutation does not occur. The answer to that question is yes. Fortunately, our cells have what's called the mismatch repair mechanism. The mismatch repair mechanism is composed of a number of proteins. The first thing these proteins are going to do is they're going to recognize if there's a problem. The reason that they're able to recognize the problem, is that when you have a mismatch in DNA it tends to distort the sugar backbone a little bit. They are going to mark the area with a cut. They are going to cut the incorrect base or mark it with a cut. The next thing that's going to happen is an exonuclease is going to remove the incorrect nucleotide. So we're going to remove the incorrect nucleotide. The next step is one of the DNA polymerases is going to insert the correct nucleotide. So we're going to pair our thymine up with adenine. The last step is a DNA ligase is going to connect the new nucleotide to the nucleotides on its sides, and also to its complementary nucleotide on the other strand. I'm actually going to just correct that distorted sugar backbone. Here's our repaired DNA. Just to clarify, the mismatch repair mechanism that we're talking about here happens after replication. The repairs done by DNA polymerase III and DNA polymerase I that we discussed before, that happens during replication or at the end of replication. One thing you might be wondering is how does the mismatch repair mechanism know to distinguish between the original parental strand and the newly synthesized strand that has the mistake on it? In other words, how does it know which base over here is correct, in our case that's the thymine, and which one is incorrect, in our case, well, it was a guanine. We know the answer to that question in bacteria. In bacteria, the parental strand will have adenines that are methylated. I'm just going to draw some methyl groups on all the adenines. That allows the mismatch repair mechanism to kind of recognize and distinguish between the original strand that has the correct base on it, and the new strand that has the incorrect base on it. But we're not quite sure how the mismatch repair mechanism in eukaryotic cells and in other prokaryotic cells knows to distinguish between the strand that has the correct nucleotide and the strand that has the incorrect nucleotide.