Discovering the structure of DNA

• DNA = Deoxyribose nucleic acid

• Made out of sugars (deoxyribose), phosphates

and nitrogen bases

Discovering the structure of DNA

• Structure was discovered in 1953 by James

Watson and Francis Crick

Discovering the structure of DNA

Rosalind Franklin’s DNA image

“Chargoff’s rule”

A = T & C = G

Cell division and DNA replication

• Cells divide

Growth, Repair, Replacement

• Before cells divide they have to double cell

structures, organelles and their genetic

information

DNA replication

Site and function of nucleic acids

• DNA • RNA• site of DNA • IN eukaryoytes: cellsDNA is found in the nucleus(chromosomes) and in

the mitochondria.• In prokaryotes: there is a single chromosome which contain

DNA .There may be also a non chromosomal DNA in the form of plasmid.

• Functions of DNA ; replications(cell division)expression of genetic information and protein synthesis(through RNA’s)

Site of RNA’s• 1. RNA’s that synthesized in the mitochondria remain within this

organelle.• 2. RNA’s that synthesized in the nucleus perform their function in the

cytoplasm.• Function of RNA’s• 1. RNA’s participate in the process of expression of genetic information

that stored in DNA (protein synthesis).• Some viruses use RNA in their its single or double stranded form as a

genetic material i.e RNA is used instead of DNA.

Steps of DNA replication• Replication folk • chain elongation reverse transcriptase • DNA repair• regulation of DNA synthesis• inhibitor of replication.

Semiconservative

• The process by which DNA is copied is called semiconservative. This mean that after replication ,each of daughter DNA mol. Will of daughter DNA mol. Will contain :

• 1. one old strand: one parental strand is conserved.

• 2.one new strand which is synthesized from free nucleotide present in the nucleus.

Cont.

• During replication , the double strand DNA mol.I(duplex) that is to be copied is separated into two strands and each is used as a template for the synthesis of a new complementary strand.

In prokaryotes

• DNA polymerase I catalyzes DNA repair.

• DNA polymerases II is unknown function.

• DNA polymerases III catalyses mostly replication of DNA.

In eukaryotes

• DNA pol- alpha catalyses replication of nuclear DNA.

• DNA pol –beta catalyses replication DNA repair.

• DNA pol gamma catalyses replication of mtochondria DNA.

• DNA pol delta responsible for leading strand of DNA replication.

Cont.

• DNA pol ε responsible for synthesis of lagging strand and repair.

A. Strand separation

• For replication: strands of DNA separated, polymerase use only single stranded DNA as template.

• IN prokaryotes.E.coli – ORIC –initiation of replication.

• IN eukaryotes :multiple site for replication along the DNA helix.

Replication folk

• As strands unwind and separate , they form the ‘V’’ shape where synthesis occur.This region is called repliction folk.

• 1. RF moves along the DNA mol. As synthesis occur.

• 2. replication of double stranded DNA is bidirectional.

Proteins responsible

• A. helix –destabilizing (HD) proteins: they bind nonenzymatically to a single stranded DNA,without interfering with the ability of the nucleotides serve as a template

• Functions:• 1wo strands separated.• Protect DNA from nuclease enzyme that

cleave single stranded DNA.

Cont.

• B. Helix unwinding proteins: also called helicase.or rep proteins.

• 1. bind single stranded DNA near the replication fork and then move into the neighbouring double stranded region.

• 2.Requires energy. 2ATP mol. Are consumed to separate each base pair.

• 3. once the strand separated destabilizing proteins binds.

Topo I and II

• “Swivels”• Prevents formation of supertwinsting

and rotation of the entire chromosome ahead of replication folk. Super twisting makes further separation more difficult and entire chromosome consume more energy.

Topoisomerase I (DNA swivelases

• They cut and rejoin a single of double helix . • This process does not require ATP as the

energy released from the cleavage (cutting ) of phosphodiester bond is reused to reseal (rejoin) the strand.

• By creating transient “nick”the DNA helix on either side of the nick is allowed to rotate at the phosphodiester bond opposite the nick ,thus relieving accumulated supertwiste.

Topoisomerase II (DNA gyrase)

• It binds to both strand of the DNA and make transient breaks in both strands of DNA helix to pass through the break and finally reseal the break .

• A negative supertwists can be introduced that allow the break unwinding of the DNA double helix.

Formation of RNA primer

• 1. polymerase III is unable to assemble the first few nucleotides of a new strand using the parent DNA strand as a template.

• 2.This assembly require RNA primer:• A. a short fragment of RNA . 10

nucleotides.• B.Complementary and antiparallel to the

DNA strand.

Cont.

• C. free -OH group at 3’end . This Oh serves as a the acceptor of the first nucleotide from DNA polymerase III.

• 3.synthesis of RNA primer requires primosome which is a complex of an protein called :RNA pol and protein called DNA B protein.

• Primosome binds with single stranded DNA and enable the initiation of synthesis of RNA primer.

Cont.

• RNA primer is later removed.

Synthesis of new DNA

• The substrate of DNA are: dATP,dGTP,dTTp,and dCTP. If one of four nucleotide is not available , DNA synthesis will blocked.

• Using the free 3ÓH group of the RNA primer as the acceptor of the first nucleotide , DNA polymerase III begins to add subsequent nucleotide.

Chain elongation

• DNA pol lII moves along the template strand , substrate nucleotide pair with the pairing rule. A=T, G=C,thus complementary to the parent strand.

• New strand runs in 5’-3’ direction while template strand runs 3’-5’. The daughter strand chain must grow in opposite directions, one towards replication fork and the other away from it.

Cont.

• Leading strand is the strand that being copied in the direction towards replication fork . It is synthesized almost continuously

• Lagging strand : is the strand being copied in the direction away from replication fork . It is synthesized discontinuously by forming small fragment of DNA called : OKAZAKI fragments.

• They are joined to become a single , continuous strand.

Excision of RNA primers and their replacement with DNA

• 1. DNA polymerase III continues to synthesize DNA un till it is blocked by a fragment of the RNA primer.

• 2.The RNA primer is excised by DNA polymerase I• 3. Gaps resulting from the excised RNA primers are filled by DNA pol I.• 4. Nicks are sealed by DNA ligase.• 5. final phosphodiester linkage between the 5’ phosphate group on the

DNA chain synthesized by DNA polymerase III and 3’ hydroxyl group on the chain made by DNA polymerase I is catalyzed by DNA ligase .The energy required for this joint is provided by cleavage of ATP tp AMP and Ppi.

Reverse transcriptase

• Also called RNA dependent DNA polymerase :• DNA – RNA - protein• Retroviruses has a mechanism for reversing the first step in this flow

form RNA to DNA.• The retrovirus contain ss RNA nucleic acid and a viral enzyme called

reverse transcriptase.

Mechanism of replication

• 1.Ss RNA ds DNA• 2.This enzyme synthesize a DNA –RNA hybride mol. Using • A) RNA genome as template.• B) dATP ,dGTP and dCTP gTTP as substrates.• 3. RNA degraded by Rnase H .• The remaining DNA strand in turn serve as a template to form a double

stranded genome of the virus.• The newly synthesized viral double stranded DNA enters the nucleus of

the infected cell and can integrate by recombination into host chromosome.

• Eg: HIV(AIDS) ,hepatitis A virus and some tumor viruse.• RT are important in recombinant DNA technolongy.

DNA repair

• A)Causes of DNA damage:• Physical agent e.g x-ray , ultraviolet light.• Chemical agent e.g alkylating agent• Ionizing radiation• B) single base alteration:• 1. depurination i.e removal of purine.• 2.deamination of cytosine to uracil• 3. deamination of adenine to hypoxathine.• 4.Alkylation of base i.e addition of alkyl group.• 5. Insertion or deletion of nucleotide.• Base analog incorporation.

Two base alteration

• a. formation of thymine –thymine dimer by ultraviolet light

Cont.

• Chain break: e.g phosphodiester bonds can be broken.• Cross linkage:• A. between bases in same or opposite strands.• B. between DNA and protein molecule e.g histones

fate of damaged DNA • 1.Repaired• 2. Replaced by DNA recombination• 3. Retained : retention leads to mutation and cell death.

Mechanism of DNA repair

Excision repair : damaged only one strand e;g thymine dimers and missing base.

Repair of pyrimidine-pyrimidine dimer:1. The dimers result form covalent joining of two adjacent pyrimidine.2. Caused by uv rays3. Thymine dimers prevent DNA pol from replicating the DNA strand beyond the

site of dimer formation.4. Dimer is excised and repair:a. Uv –specific endonuclease recognises the dimer and makes a nick near it ,at

5’ endb. gap is filled by polymerase I ,in the direction of 5’ to 3’.Other strand acts as

template.c. Thymine dimer region is excised by the 5’-3’ exonuclease activity of DNA pol

I and sealed by DNA ligase.

Xeroderma pigmentosum

• It is an autosomal recessive disease, is an e.g of a defective mechanism for the repair of pyrimidine dimers in DNA.

• Absence of uv specific endonucleases require for the recognition of the dimers is the cause of this disease.

• Individuals are sensitive to uv light which causes extensive accumulation of thymine dimers in skin cells with malignant transformation.

Some of the most common symptoms are:An unusually severe sunburn after a short sun exposure. The sunburn may last for several weeks. The sunburn usually occurs during a child’s first sun exposure. development of many freckles at an early age. Irregular dark spots. Thin skin. Excessive dryness. Rough-surfaced growths (solar keratoses), and skin cancers. Eyes that are painfully sensitive to the sun and may easily become irritated, bloodshot, and clouded. Blistering or freckling on minimum sun exposure. Premature aging of skin, lips, eyes, mouth and tongue

Repair of cytosine deamination to uracil

• Abnormal uracil is recognized by glycosylase that cleaves the base .

• Endonuclease cuts the phosphodiester bond on 5’side.

• DNA pol I fills the gap.

• DNA ligase seals the breaks.

Photoreactivation or light repair

• Thymine repair• Use visible light (300-600nm) for activating specific enzyme called

photoactivating enzyme which directly cleave and correct the dimer in its place.

Recombination repair(sister strand exchange)

• In prokaryotes (E.coli) , the cell deal with DNA replication at the dimer and reinitiating it on the other side of the dimer . This leaves gap in the newly synthesized strand b.

• By sister strand exchange , the unmutated single stranded segment from homologous DNA excised form good strand (d strand) and inserted into the gap present in b strand opposite the dimer.

• The gap in d strand is next filled by polymerase I.