5. Cell division and DNA replication
• Cells divide
Growth, Repair, Replacement
• Before cells divide they have to double cell
structures, organelles and their genetic
information
7. 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)
8. 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.
9. Steps of DNA replication
• Replication folk
• chain elongation reverse transcriptase
• DNA repair
• regulation of DNA synthesis
• inhibitor of replication.
10. 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
11. 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.
12.
13. In prokaryotes
• DNA polymerase I catalyzes DNA
repair.
• DNA polymerases II is unknown
function.
• DNA polymerases III catalyses mostly
replication of DNA.
14. 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.
15. Cont.
• DNA pol ε responsible for synthesis of
lagging strand and repair.
16. 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.
17. 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.
18. 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.
19. 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.
20.
21. 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.
22. 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
23. 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.
24. 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.
25. 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
27. 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.
28. 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.
29. 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.
30. 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.
31. 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.
32. 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.
33. 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.
35. 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
36. fate of damaged DNA
• 1.Repaired
• 2. Replaced by DNA recombination
• 3. Retained : retention leads to mutation and cell death.
37. 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 rays
3. 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’ end
b. 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.
38. 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.
39.
40. 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
41. 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.
42. 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.
43. 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.
