REPLICATION OF DNA

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Restriction enzyme

• Endonuclease – cleave the nt in the middle of DNA molecule

• Exonuclease - cleave the nt from the end of DNA molecule

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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

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Replication process

• A complex process

• Ensures fidelity

• 3 important challenge

1. 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.

2. Synthesizing DNA from 5’ end to the 3’end.

3. Guarding against error in replication – ensuring the correct base is added

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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

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Semiconservative replication

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Steps involved in DNA replication

a) Identification of the origins of replication b) Unwinding (denaturation) of dsDNA to provide ssDNA

template c) Formation of the replication fork d) Initiation of DNA synthesis and elongation e) Formation of replication bubbles with ligation of the

newly synthesized DNA segments f) 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

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DNA REPLICATION IN E.COLI-FAMOUS MODEL

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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

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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

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DNA Replication Process

1. DNA helicase unwinds short segment of parent DNA

2. SSB protein stabilize the unwound parental DNA

3. A primase initiates synthesis of RNA molecule (primer) that is essential for priming DNA synthesis

4. Initiation of rep-require priming by short length of RNA (primer) (10 to 200nts)

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Template and primer

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3’-OH 5’ Template

5’ 3’-OH Primer AGCTACTGCT…….

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

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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

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• 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

DNA Replication Process

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• Okazaki fragments are 100-250nt long in eukaryotes and 1000-2000bp 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

DNA Replication Process

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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

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DNA REPLICATION IN EUKARYOTES

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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

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• 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

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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

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DNA REPLICATION IN EUKARYOTE

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

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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

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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

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Termination of replication in eukaryotes

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Lagging strand 3’ 5’

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

5’

????????

The DNA can become shorter and shorter every time replication occur

TTAGGG

Telomerase added telomere repeats to the 3’end of the new strand-serve as primer

3’ 5’

5’

3’

3’

The last segment of DNA can be synthesized- complete the replication

5’

5’ 3’

3’

3’

3’

REPLICATION ERRORS AND THEIR REPAIR

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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

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Effect of replication errors

• Mutations – permanent changes- not fx

• Prevent it to be used as the template for replication and transcription

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Types of damages to DNA

• Single base alteration

• Two base alteration

• Chain breaks

• Cross-linkage

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Cell cycle checkpoint

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

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• 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

Proof reading mechanism

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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

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Mismatched repair 1. The newly synthesized

DNA has a mismatched

2. 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

3. An exonuclease removes DNA from the red strand between proteins (damaged dna)

4. DNA Polymerase replace the removed DNA with the correct sequence

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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

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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

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Eukaryotes

• 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

• 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

Prokaryotes

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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

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