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DNA REPLICATION IN EUKARYOTES

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Salsabila Soraya

on 28 November 2013

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Transcript of DNA REPLICATION IN EUKARYOTES

DNA REPLICATION IN EUKARYOTES
BOI 104 Genetics Group 10

Members: 
⦁ Nurul Nabila Shohimi                      121652
⦁ Shanjeellia a/p Jayaprakas      121658
⦁ Naventhan a/l Ahrasan                   121619
⦁ Syakirah Aina Binti Mohd Rashid 121666
⦁ Noor Asiah Bt Mat Rahim               121625
⦁ Aziera Bt Abdul Rahim                    121583
⦁ Norzalena Bt Abd Hamid               121634
⦁ Siti Sarah Amirah Binti Hannes     121661
⦁ Yusfariza Binti Robbiai                    121679
⦁ Salsabila Soraya Rudi                      122945
⦁ Nor Liana Bt Mohd Suhir                 121630


DNA polymerase (Alpha)
-Involved in initition
-Synthesize a RNA primer
-Has no 3'-5' exonuclease activity (proofreading) or ability to remove nucleotides

DNA polymerase (Delta)
-Principal DNA polymerase in eukaryotic DNA replication
-Has 3'-5' exonuclease activity
-Synthesizes DNA on the "lagging" strand in a fragmented or discontinuous manner.


DNA polymerase

(Beta)
-DNA repair

DNA polymersase

(Gamma)
-DNA replication enzyme of mitochondria

DNA Polymerase (Epsilon)
-synthesizes DNA on the "leading" DNA strand continuously as it is pointing in the same direction as DNA unwinding by the replisome.

RNA Primase
- The requirement for a free 3' hydroxyl group is fulfilled by the RNA primers that are synthesized the RNA primer at the initiation sites by these enzymes.

DNA Ligase
- Nicks occur in the developing molecule because the RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand. The final replication product does not have any nicks because DNA ligase join the Okazaki fragments by forming a covalent phosphodiester linkage between 3'-hydroxyl and 5'-phosphate groups.

Important terms.
Proteins of DNA Replication

To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule.

⦁ DNA Helicase
- bind to the double stranded DNA, stimulating the separation of the two strands, unwind the DNA double helix by breaking the hydrogen bond between the nitrogenous base.

⦁ DNA single-stranded binding proteins(SSBP)
- bind to the DNA as a tetramer, stabilize the single-stranded structure that is generated by the action of the helicases. Replication is 100 times faster when these proteins are attached to the single-stranded DNA.


DNA Gyrase@ Topoisomerase
- catalyzes the formation of negative supercoils that is thought to aid with the unwinding process. Relieves tension to the DNA molecule by nicking and cutting a certain place in the phosphate backbone to prevent supercoiling.


DNA Replication in Eukaryotic


⦁ More complex than prokaryotic but still have some similarities like DNA helicases, DNA polymerases, primases, ligase and etc

⦁ G1 phase-many of DNA replication regulatory processes are initiated.

⦁ S phase-majority of DNA synthesis occurs.

G2 phase - repair any damage DNA or replication errors
DNA Polymerases in eukaryotic



Initiation of DNA Replication

⦁ DNA replication begins with assembly of a pre-replication complex (pre-RC). Pre-RC acts as a key intermediate in the replication initiation process.

⦁ Origin recognition complex (ORC) with help from 2 protein factors: ⦁ cell division cycle 6 protein (Cdc6) and ⦁ chromatin licensing and DNA replication factor 1 protein (Cdt1) bring ⦁ minichromosome maintenance (Mcm 2-7) complex to replication origin.

⦁ Mcm2-7 complex loads onto DNA at replication origins during G1 and unwinds DNA ahead of replicative polymerase.
cyclin-dependent kinase (CDK) / ⦁ Dbf4-dependent kinase (DDK) dependent phosphorylation of pre-replicative proteins leads to replisome assembly and origin firing.

⦁ Cdc6 and Cdt1 are no longer required and are removed or degraded.

⦁ Mcms and associated proteins, GINS and Cdc45 unwind DNA to expose template DNA. At this point, replisome assembly is completed and replication is initiated.


Processes:

⦁ Primer synthesis by DNA polymerase-α (Pol-α).

⦁ Replication factor C (RFC) displacement of DNA polymerase and recruitment proliferating cell nuclear antigen (PCNA).

⦁ Elongation by the newly recruited DNA polymerase-δ (Pol-δ).

⦁ Strand displacement by Pol-δ.

⦁ Cutting of the 5’ displaced flap by flap endonuclease 1 (Fen 1).

⦁ Sealing by DNA ligase I (Lig I)

1. As DNA is unwound for replication, it forms a replication ‘bubble’ like form.


⦁ DNA
⦁ also known as deoxyribonucleic acid – self replicating material which is present in nearly all living organisms as the main constituent of chromosomes which carries genetic information

⦁ REPLICATION :
⦁ (GT) The process of duplicating or producing an exact copy of a polynucleotide strand such as DNA.

⦁ (GN) The act or process of generating a copy

⦁ EUKARYOTES
⦁Any of the singlecelled or multicellular organisms whose cell contains a distinct, membrane-bound nucleus.

1.Polymerase (alpha): nuclear, DNA replication, no proofreading

⦁ 2.Polymerase (beta): nuclear, DNA repair, no proofreading

⦁ 3.Polymerase (gamma): mitochondria, DNA replication, proofreading

⦁ 4.Polymerase (delta): nuclear, DNA replication, proofreading

⦁ 5.Polymerase (epsilon): nuclear, DNA repair, proofreading

Prokaryotic DNA Replication Eukaryotic DNA replication
Occurs inside the cytoplasm Occurs inside the nucleus
Only one origin of replication per molecule of DNA Have many origins of replication in each chromosome

Origin of replication is about 100-200 or more nucleotides in length
Each origin of replication is formed of about 150 nucleotides

Replication occurs at one point in each chromosome
Replication occurs at several points simultaneously in each chromosome

Only one replication fork is formed

Multiple replication forks are formed simultaneously in each chromosome
Prokaryotic DNA Replication Eukaryotic DNA replication
Only have one origin of replication Has multiple origins of replication

Initiation is carried out by protein DnaA and DnaB Initiation is carried out by the Origin Recognition Complex

DNA gyrase is needed DNA gyrase is needed
Replication is very rapid Replication is very slow


2. In human DNA, replication can begin in multiple locations at the same time

4. Replication on the other strand has to happen in short chunks. This is called the ‘lagging stand’.


5. Leading strands - Helicase unwinds the DNA strands


6. As the DNA is unwound, the strand can get twisted.
7. Topoisomerase - functions in the breaking and reforming of the bonds so that the strands don’t get too twisted.

8. Single Strand Binding Protein (SSBP) – these keeps the strands from rejoining before they can be copied

13. Replication can only go from 5’ to 3’ direction, so the lagging strand is copied in fragments.

14. The key players are the same.
- DNA Polymerase epsilon and delta : copies the DNA
- RNA Primase : helps start the replication
- DNA Polymerase alpha : add RNA codes

15. Each chunk of DNA is copied and the process start over again.


12. Then, DNA Polymerase alpha comes in and replaces the RNA codes with DNA.


9. DNA Polymerase epsilon copies the DNA at a leading strand. However the DNA Polymerase delta at a lagging strand.
10. Polymerase alpha starts DNA copying by using RNA codes


16. Each fragments has gaps that must be filled in by Ligase

11. Now the DNA polymerase epsilon can start copying and at the same time DNA polymerase delta start copying at the lagging strand

3. DNA can only replicate in one direction, from 5’ to 3’. This means that replication on one strand can occur continuously. This is called leading strand.



17. Each chunk of replicated code on the lagging strand is called an Okazaki Fragment.

Simple Explanation
origins in eukaryotes
replication in prokaryotes vs eukaryotes
Elongation
1. Initiation complex is formed and the cells pass into the S phase
2. Replication on the leading and lagging strands is performed by DNA polymerase (epsilon) and DNA polymerase (delta) respectively.
3. DNA synthesis with the unwinding of the template strand by Cdc45-Mcm-GINS complex.
4. These supercoils would cause DNA replication to halt if they were not removed.
5. Topoisomerases are responsible for removing these supercoils ahead of the replication fork.
Termination
1. Due to the fact that an RNA primer is required for initiation of DNA synthesis, the lagging strand is at a disadvantage in replicating the entire chromosome.

2. While the leading strand can use a single RNA primer to extend the 5' terminus of the replicating DNA strand, multiple RNA primers are responsible for lagging strand synthesis, creating Okazaki fragments.

3. This leads to an issue due to the fact that DNA polymerase is only able to add to the 3' end of the DNA strand.

4. The 3'-5' action of DNA polymerase along the parent strand leaves a short single-stranded DNA (ssDNA) region at the 3' end of the parent strand when the Okazaki fragments have been repaired.
epsilon
epsilon, by the way...
alpha
Polymerase Alpha
1. What is proofreading?

A: It is an error-correcting process, where an incorrect base pair is recognized by an enzyme, hence changing the base pair by re-inserting the correct base and replication can continue.

2. What is the function or benefit of DNA replication in eukaryotes?

A: It is vital for DNA to be copied in order for cell to function well, since dna is the genetic material which makes up the identity of the cell

3. What is the difference between positive and negative supercoil?

A: Positive supercoiling - one direction result from overwinding Negative supercoiling - other direction as an example unwind a rope and release the tension on it

4. Why is DNA replication in eukaryotes is slower than DNA replication in prokaryotes?

A: Eukaryotes have specific sites at which replication is originated. However, because eukaryotic cells contain much more DNA than bacteria (humans have approximately 1500 times as much DNA as E.coli), and the rate of elongation during DNA replication in bacteria is about 500 nucleotides per second, while in humans, the rate of elongation is only about 50 nucleotides per second, hence the DNA replication in eukaryotes is slower than that in prokaryotes.

Question and Answer
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