Dna replication 101

2. 1. DNA Replication
2. DNA Transcription
3. Genomic Technology and Bioinformatics

3. Chemistry and Metabolism
of Nucleic Acids

4. Replication
Transcription
Translation
DNA
RNA
PROTEIN

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6.  Early scientists thought protein was the cell’s
hereditary material because it was more
complex than DNA
 Proteins were composed of 20 different
amino acids in long polypeptide chains
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7.  Adenine must pair with Thymine
 Guanine must pair with Cytosine
 The bases form weak hydrogen
bonds
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G C
T A

8.  Rosalind Franklin took
diffraction x-ray
photographs of DNA
crystals
 In the 1950’s, Watson &
Crick built the first model
of DNA using Franklin’s
x-rays
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12.  Two strands coiled called
a double helix
 Sides made of a pentose
sugar Deoxyribose bonded
to phosphate (PO4) groups
by phosphodiester bonds
 Center made of nitrogen
bases bonded together by
weak hydrogen bonds
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13.  A gene is the sequence of nucleotides
within a portion of DNA that codes for a
peptide or a functional RNA
 Sum of all genes = genome

14.  DNA has to be copied
before a cell divides
 DNA is copied during the S
or synthesis phase of
interphase
 New cells will need identical
DNA strands
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15.  S phase during interphase of the
cell cycle
 Nucleus of eukaryotes
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Mitosis
-prophase
-metaphase
-anaphase
-telophase
G1 G2
S
phase
interphase
DNA replication takes
place in the S phase.

16. • Replication has to be extremely accurate:
• 1 error/million bp leads to 6400 mistakes every
time a cell divides, which would be catastrophic.
• Replication also takes place at high speed:
• E. coli replicates its DNA at a rate of 1000
nucleotides/second.

17. • Conservative replication model
• Dispersive replication model
• Semiconservative replication
Proposed DNA Replication Models

19.  Semiconservative
 Daughter DNA is a
double helix with 1
parent strand and 1
new strand
 Found that 1 strand
serves as the template
for new strand

20. • Theta replication: circular DNA, E. coli;
single origin of replication forming a
replication fork, usually a bidirectional
replication
• Rolling-circle replication: virus, single
origin of replication
• Linear Eukaryotic Replication: Eukaryotic
cells; thousands of origins.
Modes of Replication

21. • Requirements of replication:
• A template strand
• Raw material: nucleotides
• Enzymes and other proteins

22.  Each strand of the parent DNA is used as a
template to make the new daughter strand
 DNA replication makes 2 new complete double
helices each with 1 old and 1 new strand

23.  Site where replication
begins
◦ 1 in E. coli
◦ 1,000s in human
 Strands are separated to
allow replication
machinery contact with
the DNA
◦ Many A-T base pairs
because easier to break 2
H-bonds that 3 H-bonds
 Note anti-parallel chains

24.  Begins at Origins of Replication
 Two strands open forming Replication
Forks (Y-shaped region)
 New strands grow at the forks
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Replication
Fork
Parental DNA Molecule
3’
5’
3’
5’

25.  Before new DNA strands can form,
there must be RNA primers present
to start the addition of new
nucleotides
 Primase is the enzyme that
synthesizes the RNA Primer
 DNA polymerase can then add the
new nucleotides
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26.  Enzyme Topoisomerase attaches to the
2 forks of the bubble to relieve stress
on the DNA molecule as it separates
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Enzyme
DNA
Enzyme

27.  An enzyme that
catalyzes the addition
of a nucleotide to the
growing DNA chain
 Nucleotide enters as a
nucleotide tri-PO4
 3’–OH of sugar attacks
first phosphate of tri-
PO4 bond on the 5’ C of
the new nucleotide
◦ releasing pyrophosphate
(PPi) + energy

28.  Bidirectional synthesis of the DNA double
helix
 Corrects mistaken base pairings
 Requires an established polymer (small RNA
primer) before addition of more nucleotides
 Other proteins and enzymes necessary

29.  DNA polymerase can only ADD nucleotides to
a growing polymer
 Another enzyme, primase, synthesizes a short
RNA chain called a primer
◦ DNA/RNA hybrid for this short stretch
◦ Base pairing rules followed (BUT A-U)
◦ Later removed, replaced by DNA and the backbone is
sealed (ligated)

30.  Actually how DNA is synthesized
◦ Simple addition of nucleotides along one strand,
as expected
 Called the leading strand
 DNA polymerase reads 3’  5’ along the leading
strand from the RNA primer
 Synthesis proceeds 5’  3’ with respect to the new
daughter strand
 Remember how the nucleotides are
added!!!!! 5’  3’

31.  Actually how DNA is synthesized
◦ Other daughter strand is also synthesized 5’3’
because that is only way that DNA can be
assembled
◦ However the template is also being read 5’3’
 Compensate for this by feeding the DNA strand
through the polymerase, and primers and make many
short segments that are later joined (ligated) together
◦ Called the lagging strand

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 The Leading Strand is synthesized as
a single strand from the point of
origin toward the opening replication
fork
RNA
Primer
DNA Polymerase
Nucleotides
3’
5’
5’
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 The Lagging Strand is synthesized
discontinuously against overall direction of
replication
 This strand is made in MANY short segments
It is replicated from the replication fork
toward the origin
RNA Primer
Leading Strand
DNA Polymerase
5’
5’
3’
3’
Lagging Strand
5’
5’
3’
3’
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34. • Direction of replication:
• Leading strand: undergoes continuous
replication
• Lagging strand: undergoes
discontinuous replication
• Okazaki fragment: the discontinuously
synthesized short DNA fragments
forming the lagging strand

35.  Other enzymes needed to excise (remove)
the primers
◦ Repair polymerase – replaces RNA with DNA
◦ DNA ligase – seals the sugar-phosphate backbone
by creating phosphodiester bond
 Requires Mg2+ and ATP

37.  Base pairing rules must be maintained
◦ Mistake = genome mutation, may have
consequence on daughter cells
 Only correct pairings fit in the polymerase
active site
 If wrong nucleotide is included
◦ Polymerase uses its proofreading ability to
cleave the phosphodiester bond of improper
nucleotide
 Activity 3’  5’
◦ And then adds correct nucleotide and proceeds
down the chain again in the 5’  3’ direction

38.  Helicase opens double helix and helps it
uncoil
 Single-strand binding proteins (SSBP)
keep strands separated – large amount of
this protein required
 Sliding clamp
◦ Subunit of polymerase
◦ Helps polymerase slide along strand
 All are coordinated with one another to
produce the growing DNA strand (protein
machine)

40. • Eukaryotic DNA polymerase
• DNA polymerase a- acts like Primase to initiate
• DNA polymerase d- replicates lagging strand
• DNA polymerase e- replicates leading strand

41.  Different enzymes
recognize, excise
different mistakes
 DNA polymerase
synthesizes proper
strand
 DNA ligase joins
new fragment with
the polymer

42. The important differences between prokaryotic and
eukaryotic replication:

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 Chemicals & ultraviolet radiation
damage the DNA in our body cells
 Cells must continuously repair
DAMAGED DNA
 Excision repair occurs when any of
over 50 repair enzymes remove
damaged parts of DNA
 DNA polymerase and DNA ligase
replace and bond the new nucleotides
together
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