DNA Replication
What are the specific activities of DNA polymerase 1?
3'-5' nucleuase activity is for proof reading function so that If polymerae adds wrong base, it can recgonise and remove it at that point These DNA polymerase enzymes have multiple activities and differne plymerase enxymes have a different set/combination of these activites. Main activity - polymerization making growing DNA chain. Breakdown mode - exonuclease activities in polymerase enzymes. There are two types of exonuclease activites - 3-->5 and 5-->3' DNA polymerase 1 in Ecoli has all three of those activirs, the polymerase activity and both exonuclease activities. There are two types of exonuclease activities. 3-->5' exonuclease function is a proof reading activity so that the exonuclease can correct incorrect nucleotides added to wrong chain. 5-3' exonuclease acitivty (same direction as the synthesis) so can't correct mistakes in growing chain, it is for correcting mistakes in other part of the DNA e.g. breaks and nicks in the strand, or removing RNA primers. It will exice nucleotides from single stranded nicks or where Rna primer has been layed down for RNA rpimer itself, usually on lagigng strand.
What is the characteristics of the starting sequence at origin of replicaiton?
At the beginning there needs to be an origin of replicaiton. Without telling you anything about these sequences where orign of replicaiton arei n bacterial and eukaryotic Dna. Based on knowledge of DNA structure, you might be able to prefict the properties of the sequences of origin of replication. - AT rich so easier to break apart the strands because there are less hydrogen bonds there For intiiation of replication to take place, in E COLI we start at Ori C which is an AT rich segment. There are these specialised proteins called DNA A proteins that bind to those AT-rich sequence and they elp break those bonds and make that part of the DNA more unstabel and open up for the dNA polymerase to gain access there.
What is the function of the sliding clampm(beta subunit)
Beta subunit - make the pol 3 holoenzyme to be highly processive over 5000 residues. If those sliding clamps shown in red and ywllow weren't present the processivity of the enzyme would reduce to about 12 residues compared to over 5000 nucleotides due to the presence of the beta subunit The beta subunit forms a ring round the DNA that functions as a sliding clamo that can move along it thereby keeping the Pol3 holoenzyme from dissociating from the template Without the clamp the core enzyme would probably dissociate after replicating only 12 residues
Why is DNA replication directional?
DNA chains are extended only in the 5'->3' direction because nearly all known DNA polymerases can only add a nucleotide to the free 3'-OH group of a base-paired polynucleotide
What are the requirements of DNA replication?
DNA polymerase to add nucleotides The template strand to copy Primer with free 3-OH group All four dNTPs and Mg2+ Origin of replication Helicase DNA Gyrase SS DNA binding proteins Primase DNA ligase
When is DNA replicated?
DNA replication in any organism is not occurring all the time. In a particular cell, whether its eukaryotic or bacterial, DNA replication is not necessarily occurring all the time because there are phases of the cell cycle that are slightly different for bacteria and eukaryotess. The phases of a cell cylce for eukaryotic cells (on the left). In eukaryotic example we got the cell cycle largely divided into cell division phase (mitosis) and then this interphase divided into parts, g1 and g2 phases and then this S phase. Just a reminder that DNA replication will only occur in the S-phase (for synthesis) in the eukaryotic cell. That's when DNA replication will occur in cell cycle.
What is the central dogma of molecular biology?
Dna goes to RNA that goes to Protein
What are the functions of DNA Polymerase 1?
Dna polymerase 1 has all three activites, both exonuclease activities and synthesis activities. It can remove Rna primers and fill in the gaps of where the Rna primer was with the polymerase synthesis activity. If you have Rna primer in freen, there will always be a break in phosphodiester backbone because the polymerase aking the Dna up to that Rna primer can't make up that last bit so what happens is DNA polymerase 1 comes in and removes the Rna primer grom this process, polymersises a new strand to fill in the gap that's left in the end and this is sealed by DNA ligase. That little nick was important to begin with because that's what is recognised by DNA polymerase 1 as the starting point to know to remove that part of the DNA and know to fill it in with Dna insteado f RNA. The ligase requires energy in the form of ATP.
What is meant by fidelity of DNA replication? What are the factors contributing to the accuracy of replication?
Dna polymerase can accidentally add wrong base We need balanced nucleotide levels because if we didn't the polymerae wiould stall because it wouldn't be abel to find next nucleotide to add to it Polymerase mechanism interacts with 3' OH and is part of how enzyme can be acurateb ecause it has specific way of creating the reaction and adding the new nucleotide in a certain drection with polarity Polymerase proof reading mechanisms are part of mechanisms of dealing with potential proof reading mechanisms DNA repair enzymes which can separately detect a mistake in DNA and repair it
How does DNA replication begin? How does it continue?
Double stranded circular chromosme of a bacterium. Two strands of DNA in the chronosome are different colours. If those two circles of Dna were loosley connected, ebfreo dNA replication takes place you need to peel apart those two circular strands. The green strand is being puuled apart form the pruple one (the other) at what we call the Ori - the origin of replication where there is ap articular sequence. It is that sequence that is recognised that can melt and open up and we will talk about the propeorties of that sequence in another lectur.e there are two resulting replicaiton forks where DNA is separated from its two strands. Then you have two sets of replication machinery moving down those two strands as the strands are separated, and we are copying the DNA at the same time in those two directions, anti clcokwise or cklockwise On the right is the electromicrograph image of a piece of DNA from an E Coli It's showing the exact process as seen on the schematic on the left, where two strands are separated at origin of replicaiton
What is Nick Translation?
During replication, RNA primers are removed and replaced with DNA through the DNA Pol 1 - catalysed Nick Translation process Nicks are sealed by the action of the enzyme DNA ligase (Need ATP or NAD+ hydrolysis)
Provide an overview of the DNA replicaiton properties
Enzyme: The main enzyme is called DNA polymerase. It's actually called a DNA directed DNA polymerase. That's because sometimes you can get a DNA polymerase that instead of using a DNA template it uses an RNA template. So need to specify that this is using a DNA template to copy and therefore called a dNA directed DNA polymerase Template Strand: If we are copying Dna the template DNA is required. For this enzyme the DNA template needs to be in a single stranded stat,e and as we know in native state it exists in double stranded molecule so there needs to be some processes to make the double stranded molecule into a signle stranded template. Mechanism: We also look at the actual biochemical mechanism of aciton of the enzye (as cheistry as we get in the lectures) will look at how the bond is formed between the nucleotides, between the building blocks in resulting DNA molecule. Simplest way to describe this mechanism is it's a nucleophilic attakc of the 3' OH group of the growing DNA chain. It's that free 3' OH ion the growing chain that nucleophilically attacks an incoming phosphate group of the building blocks coming in. we call it specifically a nucleoside triphosphate. Driving Force: Something important to rememebr is the proces sof making a DNA strand is quite reversible so the process that the polymerase carries out adding nucleotides again and again can actually be reversed and start removgin them. We don't want that to happen, because role of polymerae is it's going to make the Dna for us. Need a driving force so that reaction remains in the synthesis direction rather than removal of nucleotides.
Describe DNA replication in Eukaryotes
Going to touch on eukaryotic Dna replication They have a lot more Dna to replicate than bacterium and its more complicated and is linear in a different format. Takes a lot logner to replicate. There are different types of eukaryotic Dna. There is also DNA outside the nucleus of cell, such as in mitochondria and chorloplasts, so there are different modes of replciation for the different type s of DNA. There is a broad variety of DNA polymerases for eukaryotic cells invovled in the different modes of replicaiton, alpha, beta, gamma, delta and epsilon
What are the 7 proteins involved in DNA replicaiton and what are their functions?
Helicase and DNA gyrase both are involved in the unwinding of the strands. We need a single straded template for synthesis and the DNA is double stranded to begin with. Helicase begins the unwinding process and the gyrase makes the process easier by removing the tenssion in the helical structure as it becomes unwound. Imagine if you wound two ropes together very tightly in a coil and then try unwind them it would be a funny tension that was achieved inhte strands due to the nature of them twined together and the Dna gyrase creates nicks in the strand to help relieve that tenssion. The single stranded binding proteins help to keep the DNA unwound because its very easy for the two strands to reassociate again. Primase will synthesize the RNA primer that is required to provide a free 3' OH for the DNA polymerase. In E Coli - first DNA replication process we were talking about there are two main enzymes required in the synthesis process. These are DNA polymerase 3 and DNA polymerase 1. DNA polymerase 3 catalyzes most of the elongationo f the strand. It catalyzes the blouk of Dna synthesis. DNA polymerase 1 has a special funciton of removing those RNA primers and dilling in those gaps with the polymerase activity. DNA ligase goes in and where there are gaps left from the okazaki fragments, because DNA polymerase 1 can't complete that link, the DNA ligase goes in and will seal those gaps and willl close the photsphodiester bond or gap between the strands.
What is the process of replication for the lagging strand?
Here is our double stranded molecule with red strand and blue strands as the parental strands. The replication fork is opened up to separate the strand. The motion of replcation is the left to the right. The top leading strand is being replicated in the 5'-3' direction which is the same direction as overall motion of replicaiton fork. The other strand running in the opposite direction is instead syntehsized idiscontinuously in okizaki fragments but still rnning in 5'-3' direciton as it's the only direction polymerase can synthesize them in. the reason its doing it in little chunks is because over all it needs to keep moving in this direction.
What is the replisome? What is it made up of and what is its function?
In ecoli we call this multi=protein parts that work together the replisome. It will catalyse the snthesis of both the leading and lagging strands. We often show the replicaiton process as one DNA polymerase but there are actually two DNA polymerases because we have one copying one strand and one copying the other strand. There are always two DNA polymerases working along the same two strands in the parental Dna. Even though this yellow circle here is indicating the replisome, inside that is going to be two DNA polymerases. It contains two polumerase 3's because we are talking about process in ECOLI which is DNA polymerase 3 that synthesise the bulk of Dna replicaiotn. For the leading strand to be synthesized in red here as the fork opens at the top we can see the direction of synthesis for the leading strand is in the direciton we would expect for dhna polymerase 5'3'. Because the direction of syntehsis for the lagign strand is running in the opposite direction 3-5' we cant have the same arrangement. What happensinstead is we have a looping of the lagging template strand, and allows it to be oriented in the same direction as would the template of the leading strand, but it means as the replication fork opens, the dna polymerase can't stay attached to that strand forever because it's trying to synthesize in the opposite direction so it synthesizes the cunks (okazaki dragments)
How does the polymerase 'know' which nucleotide to add next?
Incoming nucleotides are selected by their ability to form Watson-Crick base pairs with the template DNA. Like a lock and key structure.
What do we know the current model of replication to be? Why do we know this and what were supports for the other models?
Not to say either the conservative or dispersive models were ridiculous in any way - were definitely feasible. Experimentation confirmed it is the semi-conservaitve model is the way in which DNA is replicated. There were various experiments performed by scientists to prove this and there is a famous one perfomred with labeled DNA to show and give evidence that it was semiconservative model.
What is the role of DNA gyrase and DNA helicase?
Now we have helicase and Gyrase to open up the strand further and create two replicaiton forks proceding in opposite directions starting from that origin of replication. Helicase is a hexomeric complex - can see in ecample of structure from a bacteriophade, each of the six subunits has a different colour, why it is called hexomeric (6 subunits). Helicase is also known as DNA B and there was a sequence in the ordering of these proteins bassed on when they were discovered. How helicase works is the DNA becomes threaded htrough the cnetre of the helicae and it moves along a single strand and can break the hydrogen bonds between the to strands. In order to d othat it needs a lot of energy to break those bonds and therefore needs a lot of ATP for the hydrolysis. DNA gyrase is another enzymes that helps relieve the tension in the unwinding helix. It's an enzyme that is a part of a family of type 2 topoisomerases
What is the role of DNA single stranded binding proteins?
Once the strands are open and exposed its very easy for them to reanneal and become reassociated and that's double stranded Dna gain. The other problem is when DNA strands are exposed and become single stranded they become vulnerable to attack from nucleases. To help with those two different potenital issues, single stranded binding proteins will bind and coat the newly opened single strands of DNA at the replication fork. They have multiple functions. They help prevent that single stranded DNA from reasnealling aagain and wil also prevent them from forming secondary structures, sometimes you can have one strand of RNA or DNA associating with itself if there is complimentary pairing, and forming a secondary structure. It also protects those single strands from attack by nucleases. We still need access to the Dna so what happens. When the replicaiton machinery is associating with the single strands at replicaiton fork it can knock off the single stranded binding proteins and remove them as the DNA polymerases get up to them, as they reach them on the strands. They are not the barrier, they can be removed and knocked off by the Dna polymerase.
How do E. Coli terminate DNA replication?
One of the main methods bacteria use to terminate replication. Bacterial chromosome is cricular piece of double strand DNA. We start from origin of replicaiton. When that opens there is a replication fork that forms wit the strands running in opposite directions and they must meet somewhere at the other side. There are terminal regions which are thes special sites caleld terminaotr sites. They are all labeleld with different letter of the alphabet. There are several on one side on the opposite side of the ciruclar piece of Dna and some on the other side. They are special sequences that act as one way valves. Tey allow tge DNA replication machinery to move through in one direction only but once the machienry traveling in this direction, once it reaches the other valve on halfway point it will stop at this point and pause until the machinery travelling in the poosite direction, it will meet up with that paused machinery sometimes the machinery can travel through one or two before it stops, which is why they think there are multiple terminaotr sites. Tries to maintain integrity of replicated Dna cause in theory the two units sohoudl meet up at the other side halfway as a safety mechanism. Need the help of anotherp toein called tus protein. Tus stands for terminater unitlization substance. There is a diagram of the tus protein with its two domains in blue and green wrapped around Dna with it's ter sites.
Where does DNA replication begin?
One of the requirements of replicaiton is an origin or starting point of replicaiton to take place An origin of replicaiton is a location of DNA in any organism where DNA replicaiton starts and takes place. In abcteria they have a circular chromosome. They have a single origin of replication and their speed of which replicaiton takes place is quite fast - about 1000 bp per second. In eukaryotes we have a lot more DNA to replicate, instead of one rigin of replicaiton we have many origins. We also have more complicated repair and checking mechanism so our replicaiton doesn't happen as fast either, usually about 10-100bp in comaprison
Describe origins of replication in eukaryotes
Origins of replication are different in bacteria and eukaryotes In bacteria there is one origin of replicaiotn, in the eukaryotic cells, with long linear chromoeomse, there are multiple origins. Each of these will open up, and just like the single oriigin of replciaiton in bacterial chromsome you will have two replication machinery units traveling in opposite directions until gradually and eventually they meet up. They have a lot more Dna to replicate than bacterium and its more complicated and is linear in a different format. Takes a lot logner to replicate so need multiple origin sites
What properties do the E Coli polymerases have?
Pol 1 - all , Pol 2 - only polymerization and proof reading (exonuclease 3'->5')
What are the different activities that DNA polymerase can have?
Polymerase can add nucleotides in a 5' -> 3' direction (DNA polymerase 3) Exonuclease activity 3' -> 5' direction, removing nucleotides from a 3 to 5' direction (proof reading) Exonuclease activity removing nucleotides in a 5' -> 3' direction (removing primers)
If DNA polymerase can only extend in 5'-3' direction then how can both anti-parallel strands of duplex DNA be replicated simultaneously at an advncing replication fork?
Repliaiton is classified as semi-discontinous. DNA polymerae machinery and various components allow the process to happen smoothy and efficiently using a method. A scientist called okazaki helped discover this semi discontinuous model of replicaiton whereby one of the DNA strands is copied in a continuous maner and the complimetnary strand is not, and is copied in discontinous manner. We jhave two strands caleld a leading strand and lagging strand The leading strand is syntehsized continously in the 5'-3' direction in a simple manner The lagging strand is copied discontinuously in a series of fradments which are called okazaki fragments. There are a seris of these fragments that run in the 5'-3' direction but have gaps between them that need to be filledi n final strand.
What is the structure of DNA polymerase 1 and how does it relate to its function?
Structure of DNA polymerase 1 in E Coli. There is a Klenow fragment (shown on the left). This enzhyme can be broken into two main domains. Sometimes useful to just use the klenow domain because you have completely removed one of it's enzymatic activites, and in this csae it's it's 5->3' exonuclease activity for primer removal. This shows you how the differnet enzymatic acitvieis (the 3) are in different locaitons of the enzyme. In the klenow fragment there is the eynthesis region andi in pink and green is the double strand DNA molecule and that's the strand being copied and therefore theres a new strand being synthesized in the region where that polymerase activity is. The three to 5 prime exonuclease ativity also proofreading is also located up there where the synthesis is going on. That makes sense because as you are synthesizing the DNA strand you want that 3'-5' exonuclease activity to remove any mistakes. But the primer removal 5'-3' exonuclease activity is located in this other region down here and that's the region that breaks off when we are only working with the klenow fragment.
What is the DNA Polymerase 3 Holoenzyme?
The DNA polymerase 3 holoenzyme contans core enzyme with 5'-3' polymerase activity and the 3-5' nuclease activity for proof readin.g it has a clamp loader to help switch from syntheszing a primer to synthesizing DNA. Sliding clamp are shown as loops which help keep all the machinery attached or associated with the DNA otherwise it would detach and not be associated and not be able to synthesie continuously without coming away from the tmeplate. We use the word processive to describe how closely and how lon the polymerase will stay associated with the dNA and synthesie a new strand. If a DNA polymerase enzyme is highly processive it means it stays associated with the DNA template for al ong time and will synthesize a long strand of DNA before it becomes detached. The sliding clamps help to loop the DNA polymerase onto the DNA like shower rings help to keep curtain on the rail. Here where you got a circle and second cicrle called core those are the two polymerases there in the holoenzyme.
What is the primosome? What is it made up of and what is its function?
The primosome is not a single thing or very defined combination of things. The primosome is largely reffering to how we intiatie dna synthesis and the certain cponents of all the dna replicaiton machinery involved in initiation process. The two main features of the Dna replicaiton machinery that are invovled in this initiation process are the helicase opening up the stands and the primase laying down the Rna primers to allow polymerase to start the process. There are also subunits involved in this process that can be included in the primosome. It's a few things that work together for same funciton, not a physical unit. In E Coli replication is mediation by this primosome that refers to all the different components, mainly the helicase and the primase. Primase is often referred to as DNA G. Primase will catalyse the synthesis of short RNA strand usually 5-15 nucleotides in length. The primer is providing the 3' OH so DNA can recognise it and continue extending the strand with DNA nucleotides. These elements of replicaiton machinery are propelled in the direction of the replication fork in the 5'-3' direction of template for lagging strand. The primosome looks like it is reversing its direction in order to synthesise the RNA strands for the FINISH THIS SENTENCE?
What were the different proposed models of replication of DNA?
There were a few models of how this processes occurred. These were conservative, semi conservative and dispersive. In conservative model they say - When parent model is replicated we have the original parent molecule still intact and then we have a completely new DNA molecule that has two new strands of DNA that will be the same as the parent but not the actual parent. That process will then go on for another generation...etc. Semi conservaitve - the fact the two strands are complimentary, maybe they separate completely and after one strand of DNA replication you have a combination/hybrid of the old parental strand and a new strand, and the other molecule will be the other parental strand with a new strand that was complimentary to it Dispersive - maybe its random and the enzyme isn't that ordered and strategic and the resulting molecules are bits and pieces of the parental DNA and the new DNA. Maybe arbitrary replication.
What is the role of primers in DNA synthesis? Particulalry in okazaki fragments?
This primer is requred because DNA polymerase because DNA polymerase can only synthesize from a 3' OH end. T's a bit like the questionm which came first the chicken or the egg, if dna needs to be eynthesied by polymerae using a template strand but it also needs a starting point, how does it get the starting point if it wasn't there and we are just starting from scratch? Answer is given to scientists when examining different stages of DNA replicaiton, and one of these stages they noticed in the resulting DNA strand of DNA replicaiton there was some RNA in there and they couldn't figure out hwo the RNA got in the strand. It was actually part of the okazaki fragments, but didn't stay in there all the time. Gave a hint to the starting point of DNA replicaiton. The DNA polymerae needed a starting point, and at the starting point wasn't DNA at all, it was an RNA primer that also had a 3' OH that polumerae could interact with and start the repicaiton to make complimentary strand. In an ecoli these RNA primers are synthesized by an enzyme called primase and it's very siilar in eukaryotes. When we look at semi-discontinuous model of DNA replicaiton ,need one RNA primer in strand synthesized continously, but you would need multueple for the okazaki fragmentsi n the lagging strands. At what point does the RNA disappear from the resulting DNA after replicaiton, we recognise the RNA and gaps between the RNA primers and synthesized DNA and enzymes recognise those gaps and add DNA in those gaps at end of DNA replicaiton.
What is the basic reaction of DNA replication?
Top of the slide is what we need to get process started, parental dna and nucleotides and that when we combine them with all the meterials needed (list of ten) we will get the progeny DNA (resulting DNA) and the pyrophosphate If we get template DNA strand (to be copied) that is running in the direction of 3' to 5', up the top we have newly replicated strand complimentary to remplate strand running in opposite direction (5' - 3') it is the end of the growing chain here where the C is tht you will have a free 3' hydroxyl molecule and it's that free 3' hydroxyl that will carry out the nucleophilic attack on the phosphate group o the incoming nucleoside triphosphate. The DNA polymerase will carry out the nucleophilic atack reaciton and you will find new C encoproated into growing chian and it's the release of the pyrophosphate (the two phosphates on the nucleoside that aren't needed). The sugar phosphate backbone of Dna is only one phosphate group per nucleotide.
What is the driving force for DNA replication?
Way we do that is thankfully we have an enzyme present that removes one of the products of DNA replicaiton, and from removing the products we have driching force for the synthesis. One of the products formed are two phosphates joined together called pyrophosphate. There is an enzyme present called pyrophosphatase that will readily degrade or hydolize that. By removing the product it is less likely the procedure will go in reverse.
How did the structure of DNA tell us about how it was replicated?
When we determined the structure of DNA that gave us hints to how the genetic material was replicatedi n the organism. Recall DNA is double stranded helix with complimentary bases that meet with one another in the core of the helical molecule. It has not escaped our attention that this pairing immediately suggests a possible copying mechanism for the genetic material - watson and crick. That was largely the history how we started knowing more the process of replicaiton. Was largley based on structure of DNA. If you had one of those two strands of double DNA molecule, you could predict what the other strand would be because of complementarity of both strands. They suggested that this may be how the genetic material is copied and put into the new cells.
