DNA Replication and Polymerisation - Dr Ruddy

Question 1

Why does packing DNA around Histone proteins cause problems in eukaryotic replication and transcription?

Answer 1

The tight packing of the DNA around the Histones makes it very difficult for proteins needed in replication and transcription to bind

Question 2

What are the three forms of DNA and which one is the most common?

Answer 2

A,B and Z. B is the most common

A is found when DNA is dehydrated and Z is found where there are lots of GC base pairs in a sequence

Question 3

How did Meselson and Stahl demonstrate the semi-conservative nature of DNA replication?

Answer 3

They made DNA with a heavy isotope of nitrogen, Nitrogen 15, this was marked as heavy. As this double stranded DNA was marked as heavy then they could just follow through generations and see where the heavy strand goes. This is how they proved that DNA replication was a conducted semi-conservatively

Question 4

How do prokaryotes regulate replication?

Answer 4

E. coli uses a system if hemi methylated DNA to identify parental and new strands. Bacteria methylate their DNA on the adenine at the sequence GATC, they methylate their DNA at this sequence to determine their own DNA from foreign DNA but it also has a role in DNA replication. After one replication cycle the bacterial DNA becomes hemi methylated, one strand retains the methyl groups but the newly synthesised strand does not have any methyl groups, this is how the bacteria distinguishes from template mother strands and newly synthesised strands.
A protein called Seq A keeps the newly synthesised strand un-methylated. Seq A binds to hemi methylated DNA and blocks the access for the Dam methylase enzyme that would methylate the new strand. The role of Seq A is to keep the newly synthesised strand un-methylated for long enough for the bacteria to know that it has just synthesised a new stand. Seq A also inhibits DnaA from binding.
Eventually Seq A dissociates and then the Dam methylase can access the GATC sequence and methylate the newly synthesised strand. Once all of the Seq A molecules have dissociated then the two strands are methylated and DnaA binds to he 9-mer sequences.

Question 5

What happens once all of the Seq A molecules dissociate form the E. coli replicated DNA?

Answer 5

Dam methylase can access the GATC sequences and methylate the newly synthesised strand. When all the Seq A molecules have dissociated the two strands are fully methylated and then DnaA binds to the 9-mer sequences.

Question 6

What is the role of DnaA?

Answer 6

DnaA is a protein that is made in prokaryotes and its role is to activate the initiation of replication in bacteria around the origin of replication (ori)

Question 7

In eukaryotes what is a replicator?

Answer 7

The replicator is the bit of DNA that is going to be replicated, eukaryotes have many ori and they do not all replicate at the same time.

Question 8

Describe how replication is regulated in eukaryotes

Answer 8

Replication in eukaryotes is regulated with the cell cycle. At the phase G1 the replicator is chosen. During S phase the ori becomes activated.
G1;
Cdc6 along with the ORC (origin recognition complex) recognise the origin of replication that is to be replicated. Cdt1 is bound to Mcm helicase, through the interactions between the Cdt1 and cdc6 the Mcm helicase is loaded onto the complex to allow the replication to start. The Mcm helicase bound to the origin recognition complex is now termed the pre-replicative complex. This pre-RC is bound to the origin of replication.
S phase;
The Mcm helicase is activated by phosphorylation and when this happens the ORC is displaced by DNA polymerase. Mcm helicase unwinds the DNA and the DNA polymerase synthesises the new strands. The reason the ORC is displaced is to give the DNA polymerase access to the origin of replication.
G2;
After the completion of DNA replication the ORC is replaced back at the origin of replication

Question 9

What is the origin of replication called in prokaryotes?

Answer 9

OriC, replication occurs at two sides of a replication bubble until they meet.

Question 10

What is a replisome?

Answer 10

It is the all the proteins that come together and are involved in prokaryotic replication

Question 11

Describe the initiation of replication in prokaryotes

Answer 11

The oriC in E. coli is made up repeated sequecnes of DNA, a 9-mer is a 9 base repeated sequence and a 13-mer is a 13 base repeated sequence.
DnaA recognises the 5 9-mer sites and binds there, this binding causes the DNA in the 13-mer regions to unwind and this allows the recruitment of DnaB and DnaC. DnaB is a helicase and DnaC is a helicase loader. The helicase activity of DnaB further unwinds the DNA. The Helicase DnaB is inactive until loaded onto the DNA by the helicase loader.

Question 12

What are the initiator proteins in prokaryotes

Answer 12

Initiator proteins are proteins that bind to the OriC to allow replication to occur.

Question 13

Describe how the two newly replicated circles in prokaryotes separate

Answer 13

Immediately after replication you have two-two stranded DNA circles linked together, they are intertwined and now need to separate. Topoisomerase II nicks a double strand break and separates the two strands, this process is called decatenation. The topoisomerase nicks one DNA double strand and passes the other through the break, thus separating the two DNA circles.

Question 14

What does “Catenanes” mean?

Answer 14

Two circular linked pieces of DNA.

Question 15

Why is the function of topoisomerases really important in eukaryotes?

Answer 15

Eukaryotic DNA is much more condensed and packaged compared to prokaryotic DNA, there the topoisomerases role is especially important in freeing up sequences of DNA that need to be replicated or transcribed.

Question 16

How many strands does a topoisomerase I cut?

Answer 16

it creates one stranded cuts

Question 17

Describe the end replication problem

Answer 17

As eukaryotes have their genome arranged linearly on chromosomes, the more replications occur the more the chromosome shortens at a region called the telomere. This problem only applies to the lagging strand. The last okazaki fragment causes the issue, they are used in a backstitching mechanism and the strand is synthesised from the fragment back towards the replication bubble (this is because DNA polymerase has to synthesise in a 5’ to 3’ direction). The last okazaki fragment is destroyed like all of the others as it is an RNA primer used to allow the DNA polymerase to make the bits in between them. However the last okazaki fragment that is destroyed cannot be filled in by the next fragment in the sequence as there is not one. Each replication the chromosomes are becoming shorter and shorter, this cannot continue as eventually you will have no chromosomes left.

Question 18

Describe the solution to the end replication problem using protein priming

Answer 18

Using protein priming as opposed to an RNA primer to provide the OH group for replication solves the problem. You can use a protein which just provides the OH group which is necessary for the synthesise of DNA. This solution can be found in viruses and bacteria that have linear chromsomes.

Question 19

Describe the solution to the end replication problem using telomerase

Answer 19

The enzyme telomerase is a large riboprotein complex, it contains both RNA and protein. It is a type of DNA polymerase and it has a complementary sequence to the repeated sequences found in the telomeres of eukaryotes. This complementarity allows the telomerase to recognise the telomere sequence and bind to the 3’ end. The telomerase has reverse transcriptase activity and it extends the end of the telomere using its own sequence as a template. Once it gets to the end of its sequence it dissociates and then binds again and repeats until it has done the whole telomere sequence. The 3’ strand is always slightly longer even after 5’ extension. The okazaki fragment binds in the extended 3’ region and therefore will allow DNA polymerase to synthesise the part of the DNA that would be been missed out

Question 20

What is the Hayflick number?

Answer 20

The number of times that a cell can usually divide. Normal cells limit is about 40-50 generations, cancer cells get around this and immortalise, they have elevated telomerase activity.

Question 21

What are the 3 rules regarding DNA polymerase?

Answer 21

DNA polymerase enzyme catalyses the addition of nucleotides
A template is needed
Polymerisation occurs in the 5’ to 3’ direction

Question 22

What DNA polymerase has the highest processivity?

Answer 22

DNA polymerase III, This DNA polymerase is the most effective at binding to the origin of replication and making new daughter strands, it is the most effective polymerase at making new strands of DNA.

Question 23

What is the difference between the core of an enzyme and a holoenzyme?

Answer 23

The core of the enzyme has the capacity to do the job but the holoenzyme includes all the subunits to do it effectively.

Question 24

Describe DNA synthesis by DNA polymerase

Answer 24

DNA synthesis proceeds in the 5’ to 3’ direction. DNA polymerase requires a primer, and the enzyme adds a new nucleotide at the 3’OH group of the previous nucleotide. The OH undergoes a nucleophillic attack on the alpha phosphate and if the base pairs are correct then the reactants will be correctly aligned for the nucleophillic attack to occur. Two pyrophosphates are released and these are degraded by pyro-phosphatase into 2 organic phosphates and this is what provides most of the free energy for this mechanism to occur.

Question 25

Describe nick translation

Answer 25

Okazaki fragments are made independently and are joined together. Completion of an okazaki fragment leaves a nick between the okazaki fragment and the preceding RNA primer on the lagging strand. DNA polymerase I extends the okazaki fragment while its 5’ to 3’ exonuclease activity removes the RNA primer. This process move the nick along the lagging strand. DNA polymerase I dissociates after extending the fragment 10-12 nucleotides and then DNA ligase binds to seal the ends together.

Question 26

What enzyme makes the RNA primers used to synthesis the lagging strand

Answer 26

Primase

Question 27

Describe the synthesise of the lagging strand

Answer 27

As helicase unwinds the DNA template, Primase synthesises an RNA primer. The lagging strand polymerase completes an okazaki fragment.
When the lagging strand polymerase encounters the preceding okazaki fragment, it releases the lagging strand.
The lagging strand polymerase binds to a newly synthesised primer and begins synthesising another okazaki fragment.
The lagging strand template loops back through the replisome so that the leading strand and lagging strands are synthesised in the same direction. This model is known as the trombone model. DNA polymerase exists as a dimer during replication. Sliding clamps increase processivity. A primosome consists of primase that makes the RNA primer.

Question 28

What is the role of the beta clamp or sliding clamp?

Answer 28

Ensures that the polymerase stays in the thread of DNA and secures it, this increases the processivity of the DNA polymerase.

Question 29

What is the role of the Gamma complex?

Answer 29

This is the clamp loader, it assists the clamp to load onto the DNA correctly

Question 30

Describe the function of DNA polymerase using the right hand metaphor

Answer 30

The thumb of the enzyme does not have a catalysing role but a stabilising role. The palm region contains the catalytic site for polymerisation, the DNA comes in as a double stranded helix and in this site it is separated out into single stranded DNA. There is also a kink in the DNA template that ensures that the OH is readily accessible in the active site. When the nucleotides are aligned correctly the fingers close in and this brings the catalytic site closer to facilitate the reaction. If the wrong base pair is incorporated the rate of catalysis immediately slows down, the DNA polymerase changes conformation to shift the DNA down into another active site that is a 3’ to 5’ exonuclease and this deletes the last nucleotide that was added and then the conformation resorts itself to normal and the rate of catalysis returns to normal speed.

Question 31

Why doenst mRNA need to be proofread as much as DNA?

Answer 31

because is doesnt stay around for long periods of time like DNA does, it is degraded pretty quickly anyway.

Question 32

What are the two cations in the DNA polymerase active site and what do they do?

Answer 32

Mg is there to influence the nucleophillic reaction, this helps the nucleophillic attack on the phosphates
The other cation is for stabilising the pyrophosphates that are released - havent got a name for it

Question 33

Why cant RNA get into the DNA polymerase

Answer 33

The active site of DNA polymerase is small enough to sterically inhibit RNA access and thus ensuring that only DNA is incorporated into the replicated strand. RNA is much more abundant then DNA so this is essential to make sure that RNA doesn’t accidentally get incorporated.

Question 34

Give examples of chemicl cross linking agents and there uses

Answer 34

Anti cancer agents introduce additional crosslinks between the two strands preventing replication and being lethal to the cell.
Mustard gas cross links between G on DNA, creates an alkylated adduct, which is lethal, it has some limited applications in certain leukaemias
Cisplatin - cross linking agent between DNA so it cannot replicate, it binds between two purines
Psoralen - a tricyclic compound and another cross linking agent that is found in carrots. When it is activated by UV light it forms a monoadduct with a pyrimidine and once this is formed and the UV light slightly changes wavelength then the structure can cross link to another pyrimidine.