4. Nucleic Acids (NAs)
•Info – carrying chemical cmpds of the cell.
•Direct CHON synthesis & determine inherited
xtics of orgs.
•Two main classes:
i. RNA – stores info in ‘genetic code’.
ii. DNA - carries info to ‘protein synthesis
machinery’.
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8. NA Structure
•Nitrogenous bases: two types:
a. Purines :Adenine (A),
Guanine (G).
b. Pyrimidines : Cytosine (C),
Thymine (T) in DNA, Uracil (U)
in RNA.
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9. NA Structure
Purines:
• 6 + 5 membered
rings
• Heterocyclic
aromatic cmpds.
•Pyrimidine ring
fused with an
imidazole ring.
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10. NA Structure
Pyrimidines
• 6 membered ring.
•Heterocyclic
aromatic cmpds.
• Pyrimidine ring
with N atoms at 1
and 3.
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•Phosphate grp + pentose
sugar + nitrogenous base =
nucleotide.
•Nucleoside = nucleotide
minus phosphate grp
•Nucleotide is a monomer.
•DNA or RNA is a polymer of
nucleotides.
15. DNA Structure
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•DNA strand has A, G, C and T.
•Has no U.
•A strand has 5’ end and 3’ end.
•The pentose sugar is
deoxyribose (H, not OH at C2).
•Two strands zip up to form
double strand.
17. DNA Structure
• 1953, James Watson and
Francis Crick
•Double helix DNA model.
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18. DNA Structure
• Watson and Crick based their discovery on Rosalind Franklin and
Erwin Chagarff’s work.
• Franklin: x-ray shows DNA was double helix of even width.
• Chagarff’s rule:
• A = T
• G = C
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24. DNA Replication
•Basis for inheritance.
•Occurs in proliferating cell.
•To copy DNA & transfer
genetic info to daughter
cells.
•Occurs in S-phase of cell
cycle, prior to mitosis / cell
division.
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25. DNA Replication
•Existing DNA mlcl acts as a template.
•Two identical daughter DNA mlcls produced from
parent DNA.
•Similar, with few differences, between prokaryotes
and eukaryotes.
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29. DNA Replication Prokaryotes
•Just like in eukaryotes,
occurs in three main steps:
1. Initiation
2. Elongation
3. Termination
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Take note of all
proteins (enzymes)
And their roles
30. 1. Initiation in Prokaryotes
•Begins at Ori-C (origin of replication)
•Ori-C has:
a. Three 13 mer bands rich in A andT.
• One 13 mer is 5ʹ-GATCTXTTTATTT
-3ʹ.
• Collectively called DUE (duplex unwinding elements).
• DNA unwinding occurs here.
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31. Initiation in Prokaryotes
b. Four 9 mer bands
• One 9 mer is 5ʹ-TGTGAATAA-3ʹ.
• 9 mer bands named R1, R5, R2 and R4 boxes.
• collectively called DAR (Dna A assembly region).
• negative supercoiling of DNA occurs here.
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32. Initiation in Prokaryotes
•In between R1 and R5 is IHF (integration host
factor) binding site.
•Here, protein binding creates DNA kinks and
promote initiation.
•In between R2 and R4 is FIS (factor for inversion
stimulation).
•FIS protein binding negatively regulates replication.
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34. Initiation in Prokaryotes
• DNA A protein (with ADP or ATP) binds to R
boxes.
• DNA coils around the protein.
• This induces topological stress.
• Then denaturation of DUE occurs.
• SSB protein binds to DNA strand prior or
after helicase binding.
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36. Initiation in Prokaryotes
• SSBP prevents DNA strand reanealling.
• SSBP protects ssDNA from nucleases that cleave
phosphodiester bonds.
• By now, hydrogen bonds are exposed.
• DNA C Protein (Helicase loader) complexed to DNA B
protein (Helicase) loads helicase to DUE site.
• Helicase separates dsDNA by breaking the hydrogen
bonds.
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38. Initiation in Prokaryotes
•This forms replication bubble.
•Helicase action increases DNA supercoils distal to
replication fork.
•Topoisomerase binds to the dsDNA ahead of
replication fork.
•The nuclease domain of topoisomerase breaks one
strand of the DNA.
•Then DNA unwinds and reduce the supercoils.
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39. Initiation in Prokaryotes
•The ligase domain of topoisomerase seals nicks in
the strand.
•DNA G protein (primase) binds to ssDNA
•Primase lays down small RNA primers.
•DNA polymerase III is loaded to ssDNA at primer
site.
•Elongation can begin.
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41. Initiation in Eukaryotes
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• Pre-RC (pre-
replication
complex)
formation occurs
prior to S-phase.
• Origin is rich in A
andT.
• But why??????
Binding of ORC to origin initiates
replication
48. •We did Initiation in both prokaryotic
and eukaryotic cells
•We proceed with ELONGATION
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49. 2. Elongation
•Primase reads the ssDNA from 3ʹ to 5ʹ end.
•It synthesizes RNA nucleotides in 5ʹ to 3ʹ to
form RNA primers.
•DNA pol III needs the 3ʹ OH of RNA primer.
•DNA pol III reads the DNA strand from 3ʹ to 5ʹ
end.
•It synthesizes new DNA strand in 5ʹ to 3ʹ
direction towards replication fork.
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50. Elongation
• DNA pol III proofreads in 3ʹ to
5ʹ
• It uses its 3ʹ to 5ʹ exonuclease
activity to cut out nucleotide
and replace with the correct
one.
• This happens on leading strand.
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52. Elongation
•On the lagging strand, a multiple primers are
needed for DNA pol III.
•Such many RNA primers are called Okazaki
fragments.
•DNA Pol I cuts out RNA primers from 5ʹ to 3ʹ using
the 5ʹ to 3ʹ exonuclease activity.
•DNA Pol I reads the parent strand in 3ʹ to 5ʹ.
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53. Elongation
•Then it synthesizes nucleotides on new strand in 5ʹ
to 3ʹ.
•It proofreads the parent strand in 3ʹ to 5ʹ.
•And cuts out in 3ʹ to 5ʹ exonuclease type of fashion.
•Ligase joins the DNA sections synthesized by DNA
pol I on lagging strand.
•Finally termination can occur.
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55. 3. Termination
•In prokaryotes, forks meet on opp sides of the
circular DNA.
•Termination site A terminates counterclockwise
moving fork.
•Ter C terminates the clockwise moving fork.
•The rest are back up sites.
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56. •At termination site:
✓TUS (termination utilizing substance) protein
binds to Dna B.
✓Helicase activity is inhibited.
✓Dna B is released.
✓Termination results.
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58. Termination in Eukaryotes
•At the end of chromosome are non-
replicated telomeres.
•Telomeres do not code for any RNA.
•Multiple DNA replication shortens telomeres
up to a hayflick limit.
•Hyflick limit: max number of replication
before interfering with genes on DNA
strand.
•Thus telomeres prevent gene loss.
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60. Termination in Eukaryotes
• Telomeres have sequenceTTAGGG.
• Telomerase enzyme has RNA sequence
complementary to the telomeres.
• Thus, telomerase enzyme has AAUCCC
RNA sequence.
• High telomerase activity in stem cells.
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64. Recap: Enzymes
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Enzyme Role
DNA Polymerase I Removes primers
DNA Polymerase III Deoxynucleotide polymerization
Helicase dsDNA unwinding
Primase RNA primers synthesis
Topoisomerase / Gyrase Torsional stress relief
SSBP Premature reannealing prevention
Ligase Seals nicks in new DNA strand
65. Prokaryotic vs Eukaryotic DNA Replication
Prokaryotic DAN Replication Eukaryotic DNA Replication
Occurs inside the cytoplasm Occurs inside the nucleus
One Ori-C per replicating DNA molecule Multiple origins in each molecule
Initiation by Dna A and Dna B Initiation by ORC
Gyrase relieves torsion stress Topoisomerase II
Replication is rapid Replication is slow
Continuous process In S-phase only
Involve DNA pol I and III DNA polymerase ɑ, δ and ε are involved
Two circular chromosomes are obtained Two sister chromatids are obtained
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66. Prokaryotic vs Eukaryotic DNA Replication
•Both occur before nuclear division.
•The DNA are double-stranded.
•The replication occurs in 5’ to 3’ direction.
•In both, SSBP stabilizes the unwound DNA.
•In both, primase synthesize RNA primer.
•In both processes, replication is bi-directional.
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