1. REPLICATION OF DNA

2. Restriction enzyme
Endonuclease – cleave the nt in the middle of DNA molecule
Exonuclease - cleave the nt from the end of DNA molecule

3. The flow of genetic information
Duplication of DNA to produce a new DNA molecule with the same base sequence as original
A necessary process whenever a cell divides to produce daughter cells
Flow of genetic information
DNA –RNA-Protein

4. Replication process A complex process Ensures fidelity
3 important challenge
Separating the two strands – the double helix must be unwound if they are to be separated. At the same time, the unwound portions must be protected from the action of nucleases that prefer to attack ss DNA.
Synthesizing DNA from 5’ end to the 3’end.
Guarding against error in replication – ensuring the correct base is added

5. Types of replication Conservative
The parental strands never completely separate
After on round replication, one daughter duplex contains only parental strands and the other only daughter strands
Semi conservative
The process of unwinding, of the double helical daughter molecules – that is composed of a parental strand and a newly synthesized strand formed from the complementary strand
Theta replication – replication in a circular form -prokaryote

6. Semiconservative replication

7. Steps involved in DNA replication
Identification of the origins of replication
Unwinding (denaturation) of dsDNA to provide ssDNA template
Formation of the replication fork
Initiation of DNA synthesis and elongation
Formation of replication bubbles with ligation of the newly synthesized DNA segments
Proof reading process
DNA replication in Prokaryote and eukaryote generally involve the same steps, except in eukaryote the process is more complex due to the presence of histone complex

8. DNA REPLICATION IN E.COLI-FAMOUS MODEL

9. a) Origin of replication
Origin of replication (oriC )- spesific sites where replication starts
Sequence specific DNA binding proteins (O Protein) will bind to ori
Adjacent to ori is A+T region
Binding of O protein lead to local denaturation and unwinding of A+T region

10. b) Unwinding of DNA to form SS DNA which act as template
Interaction of O protein provide a short region of ssDNA essential for initiation of replication
This region – a template for initiation
DNA helicases helps in unwinding of DNA
DNA helices produces nicks in one strand of double helix
Single stranded binding proteins (SSB Proteins) – bind to SS each strand and stabilize the complex and prevents re-annealing

11. DNA Replication Process
DNA helicase unwinds short segment of parent DNA
SSB protein stabilize the unwound parental DNA
A primase initiates synthesis of RNA molecule (primer) that is essential for priming DNA synthesis
Initiation of rep-require priming by short length of RNA (primer) (10 to 200nts)

12. Template and primer 5’
Primer
3’-OH
AGCTACTGCT……. 5’
3’-OH
Template

13. DNA Replication Process
Priming process-nucleophilic attack by 3’-OH group of the RNA primer on the α-phoshate of the first entering nucleotide with release of pyrophosphate
Elongation-3’-OH of the newly attached nt is then free to carry out nucleophilic attack on the next entering nt

14. DNA Replication Process
DNA polymerase III begin replication by adding new complementary strand nt to the primer-producing polynucleotide chain
DNA polymerase III cannot initiate DNA synthesis ‘de novo’
Leading strand (fwd strand)-the DNA is synthesized continuously in 5’to3’ direction with the same over-all fwd direction

15. DNA Replication Process
Lagging strand – the DNA is synthesized in discontinuous manner-not directly as leading strand- DNA Pol III only add nt to 3-OH – So the system is reversed to comply to its function
As replication fork is produced, a primer is synthesized from 5’ to 3’ and DNA Pol III begin the polymerization process –producing short DNA strand – Okazaki fragments

16. DNA Replication Process
Okazaki fragments are nt long in eukaryotes and bp in prokaryotes
RNA primers will be removed by DNA Pol I (using its exonuclease activity)
Leaving primers leave a gap (at least one nt missing) -
The gap will be replaced by nt by DNA Pol I – leaving only a nick (interruption in the phosphodiester bond with no missing nts)
These nicks are sealed by DNA ligases producing a long polynucleotide chains

17. Termination of replication (prokaryote)
The 2 replication forks of E.Coli meet at terminus region (Ter)
Terminal Utilization Protein (Tus) will bind to Ter – forming Tus-Tur Complex and stop the rep fork- completing 2 interlinked circular chromosome (catenated)
DNA Topoisomerase IV separated the chromosome – segregate into daughter cells

18. DNA REPLICATION IN EUKARYOTES

19. DNA REPLICATION IN EUKARYOTE
Mostly studied - yeast
The understanding is not as much as in prokaryotes
More complicated
Multiple Ori- replicators (around 400 replicators in yeast)
Timing must be controlled to that off cell division
More proteins and enzymes involved

20. DNA REPLICATION IN EUKARYOTE
Cell growth- M, G1, S and G2
Gap 1 (G1) Cell prepare for DNA synthesis
DNA replication occur in S phase – synthetic S phase
Transition from one phase to another is controlled by cyclins proteins
The DNA is replicated once and only once

21. DNA REPLICATION IN EUKARYOTE
Cancer-causing virus (oncoviruses) and cancer inducing genes (oncogenes) – capable of disrupting the entry of mammalian cells from G1 to S – excessive production of cyclin – production in an appropriate time – abnormal cell division
Once a chromatin has been replicated, it is marked to further its replication until it again passes through mitosis

22. The DNA Polymerase Complex
E.Coli
Mammalian
Function I α
Gap filling and synthesis of lagging strand II ξ
DNA Proof reading and repair β
DNA Repair γ
Mitochondrial DNA synthesis
III δ
Processive, leading strand synthesis
In mammalian cell, the polymerase is capable of polymerizing about 100nt persecond – ten fold slower than in bacterial

23. Replication bubble
In prokaryote- genome size is around 6x106 bp – replication is completed in 30 mins (replication rate 3x105bp per min)
In eukaryote – genome size is 3x109
Replicated in the same rate – will take 150 hours to complete!
Problem is overcome by having multiple origin in chromosome and replication occur in both direction along all of the chromosome

24. Termination of replication in eukaryotes
Require a special mechanism because the DNA is linear
Leading strand -5’ end template is not a problem, because the DNA is synthesized from 5’to3’ (continuous process and require only one primer in the beginning.
Lagging strand- problem

25. Termination of replication in eukaryotes
The last RNA Primer need to be removed, the gap need to be filled with DNA, but without RNA, the DNA cannot be synthesized
Lagging strand
The DNA can become shorter and shorter every time replication occur 3’ 5’ 5’ 3’ 3’
Lagging strand 5’
???????? 3’ 5’
Telomerase added telomere repeats to the 3’end of the new strand-serve as primer 5’ 3’
TTAGGG 3’
The last segment of DNA can be synthesized- complete the replication 5’ 3’ 3’

26. REPLICATION ERRORS AND THEIR REPAIR

27. The rapid repair is needed as they may be lethal to the cell
Causes of DNA Damage
DNA damage during DNA replication can occur through:
Mis-incorporation of dntp during replication
By spontaneous deamination of bases during normal genetic functions
From X- radiation that cause nicks in the DNA
From various chemicals that interact with DNA
The rapid repair is needed as they may be lethal to the cell

28. Effect of replication errors
Mutations – permanent changes- not fx
Prevent it to be used as the template for replication and transcription

29. Types of damages to DNA Single base alteration Two base alteration
Chain breaks
Cross-linkage

30. Cell cycle checkpoint

31. Proof reading mechanism
Error can occur once in 104 to 105 base pairs
Proof reading -Removal of incorrect nt immediately after they are added to the growing DNA during the replication process
Performed by DNA Pol I
DNA Pol I have two major components –
Klenow fragment (for polymerase & proofreading activity)
5’ to 3’ repair activity

32. Proof reading mechanism
Occur at the last stage of replication, after removing of primers -Through cut and patch process
Cut – removal the DNA mistakes as it moves along the DNA
Patch – Fills in the right nt

33. Some replication errors escapes proofreading
Proof reading activity- increase the fidelity
However, there are some errors escape
These errors need to be minimized before it become permanent damaged in the DNA sequence
Performed through genome scanning by the protein MutS

34. Mismatched repair The newly synthesized DNA has a mismatched
MutH, Mut S, Mut L complex will bring the mismatch with the nearest methylation site – to identify the parent strand. The methylated strand is the parent strand
An exonuclease removes DNA from the red strand between proteins (damaged dna)
DNA Polymerase replace the removed DNA with the correct sequence

35. Base excision repair
To repair base that is damaged by oxidation or chemical modification
The damaged base is removed by DNA glycosylase –leaving AP site
AP endonuclease then removes s the sugar and PO4 from the group
Excision nuclease removes several more bases
DNA Pol I fill the gap

36. Nucleotide excision repair
For DNA damaged due to the UV or chemical effect – lead to deformed DNA structure
ABC excinuclease remove the large section of damaged DNA
DNA Pol I add new nt
DNA ligases seal the gap

37. Eukaryotes Prokaryotes
Initiation point specific but different in prokaryotes
DNA Pol – 5 types –αξβϒδ
Functional variety of DNA polymerase is specific
γ DNA polymerase In mitochondria
Β- Polymerase functions as repair enzyme
Many replication forks
Theta structure not observed
Many accessory proteins with diverse functions
Histone separation from DNA as well as unwinding takes place
Many replication bubbles
RNA/DNA as primer
Initiation point specific (ori)
DNA Polymerase- 3 types – I, II, III
DNA Pol I has diverse function
Not applicable
No repair function
Replication with few replication forks
Theta structure observed
Accessory proteins few with limited functions
Only unwinding takes place in prokaryotes
Not at all or few replication bubbles
RNA as primer

38. Summary Identification of sites of the origin of replication (ori)
Unwinding of parental DNA (dsDNA ssDNA)
Formation of replication fork
Synthesis of RNA primer, complementary to DNA template, the enzyme required is primase
Leading strand is synthesized in the 5’to 3’ direction by the enzyme DNA polymerase
Lagging strand is synthesized as Okazaki fragments
RNA pieces are removed when polymerization is complete
The gaps are filled by nt and the pieces are joined by DNA ligases