Transcript DNA & DNA Replication

DNA & DNA Replication
History
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DNA
Comprised of genes
 In non-dividing cell nucleus
as chromatin
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 Protein/DNA
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complex
Chromosomes form during
cell division
 Duplicate
to yield a full set in
daughter cell
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Nucleic acids are polymers
Monomers are called nucleotides
 Nucleotides = base + sugar + phosphate
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 Base
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= purine or pyrimidine
Purines = adenine, guanine
Pyrimidines = thymine, cytosine, uracil
 Sugar
= deoxyribose or ribose
 Phosphate, a single phosphate in DNA
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Sugar of nucleotide 1 is linked to the
phosphate of nucleotide 2 by a
phosphodiester bond
DNA is a Double Helix
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Nucleotides
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A, G, T, C
Sugar and phosphate
form the backbone
Bases lie between the
backbone
Held together by
H-bonds between the
bases
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A-T – 2 H bonds
G-C – 3 H bonds
H - Bonds
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Base-pairing rules
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AT only (AU if DNARNA hybrid)
GC only
DNA strand has
directionality – one end is
different from the other end
2 strands are anti-parallel,
run in opposite directions
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Complementarity results
Important to replication
Helical Structure
Nucleotides as Language
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We must start to think of the nucleotides –
A, G, C and T as part of a special
language – the language of genes that we
will see translated to the language of
amino acids in proteins
Genes as Information Transfer
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
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DNA Replication
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
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DNA Template
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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
Replication Origin
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Site where replication
begins
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Strands are separated to
allow replication machinery
contact with the DNA
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1 in E. coli
1,000s in human
Many A-T base pairs
because easier to break 2
H-bonds that 3 H-bonds
Note anti-parallel chains
Replication Fork
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Bidirectional movement of the DNA replication machinery
DNA Polymerase
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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 triPO4 bond on the 5’ C of
the new nucleotide
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releasing pyrophosphate
(PPi) + energy
DNA Polymerase
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
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How is DNA Synthesized?
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Original theory
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Begin adding nucleotides at origin
Add subsequent bases following pairing rules
Expect both strands to be synthesized simultaneously
This is NOT how it is accomplished
How is DNA Synthesized?
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Actually how DNA is synthesized
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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
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Remember how the nucleotides are
added!!!!! 5’  3’
How is DNA Synthesized?
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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’
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 Compensate
for this by feeding the DNA strand
through the polymerase, and primers and make
many short segments that are later joined (ligated)
together
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Called the lagging strand
DNA Replication Fork Fig 6-12
Mistakes during Replication
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Base pairing rules must be maintained
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Mistake = genome mutation, may have
consequence on daughter cells
Only correct pairings fit in the polymerase
active site
If wrong nucleotide is included
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Polymerase uses its proofreading ability to cleave
the phosphodiester bond of improper nucleotide
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Activity 3’  5’
And then adds correct nucleotide and proceeds
down the chain again in the 5’  3’ direction
Proofreading
Starting Synthesis
DNA polymerase can only ADD nucleotides
to a growing polymer
 Another enzyme, primase, synthesizes a
short RNA chain called a primer
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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)
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Primers – cont’d
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Simple addition of primer
along leading strand
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RNA primer synthesized 5’
 3’, then polymerization
with DNA
Many primers are needed
along the lagging strand
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1 primer per small
fragment of new DNA
made along the lagging
strand
Called Okazaki fragments
Removal of Primers
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Other enzymes needed to excise
(remove) the primers
Nuclease – removes the RNA primer
nucleotide by nucleotide
 Repair polymerase – replaces RNA with DNA
 DNA ligase – seals the sugar-phosphate
backbone by creating phosphodiester bond
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 Requires
Mg2+ and ATP
Other Necessary Proteins
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Helicase opens double helix and helps it
uncoil
Single-strand binding proteins (SSBP) keep
strands separated – large amount of this
protein required
Sliding clamp
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Subunit of polymerase
Helps polymerase slide along strand
All are coordinated with one another to
produce the growing DNA strand (protein
machine)
Components of the DNA Replication
Polymerase & Proteins Coordinated
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One polymerase complex apparently synthesizes
leading/lagging strands simultaneously
Even more complicated in eukaryotes
DNA Repair
For the rare mutations occurring during
replication that isn’t caught by DNA
polymerase proofreading
 For mutations occurring with daily
assault
 If no repair
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In germ (sex) cells  inherited diseases
 In somatic (regular) cells  cancer
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Effect of Mutation
Uncorrected Replication Errors
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Mismatch repair
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Enzyme complex recognizes mistake and excises
newly-synthesized strand and fills in the correct
pairing
Mismatch Repair – cont’d
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Eukaryotes “label”
the daughter strand
with nicks to
recognize the new
strand
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Separates new from
old
Depurination or Deamination
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Depurination – removal of a purine base from
the DNA strand
Deamination is the removal of an amine group
on Cytosine to yield Uracil
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Could lead to the insertion of Adenine rather than
Guanosine on next round
Chemical Modifications
Thymine Dimers
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Caused by exposure to UV light
2 adjacent thymine residues become
covalently linked
Repair
Mechanisms
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Different enzymes
recognize, excise
different mistakes
DNA polymerase
synthesizes proper
strand
DNA ligase joins new
fragment with the
polymer