Загрузил Kseniia Nazarenko

Chapter 5 & 6 - DNA & DNA Replication

Chapter 5 & 6
DNA & DNA Replication
Comprised of genes
 In non-dividing cell nucleus
as chromatin
 Protein/DNA
Chromosomes form during
cell division
 Duplicate
to yield a full set in
daughter cell
DNA is Genetic Material
From Chapter 2
Nucleic acids are polymers
Monomers are called nucleotides
 Nucleotides = base + sugar + phosphate
 Base
= purine or pyrimidine
Purines = adenine, guanine
Pyrimidines = thymine, cytosine, uracil
 Sugar
= deoxyribose or ribose
 Phosphate, a single phosphate in DNA
Sugar of nt 1 is linked to the phosphate of nt
2 by a phosphodiester bond
Panel 2-6
Chapter 2
– cont’d
DNA is a Double Helix
A, G, T, C
Sugar and phosphate
form the backbone
Bases lie between the
Held together by
H-bonds between the
A-T – 2 H bonds
G-C – 3 H bonds
H - Bonds
Base-pairing rules
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
Complementarity results
Important to replication
Helical Structure
Nucleotides as Language
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
DNA Replication
 Daughter DNA is a
double helix with 1
parent strand and 1
new strand
 Found that 1 strand
serves as the
template for new
DNA Template
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
Site where replication
Strands are separated to
allow replication machinery
contact with the DNA
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
Bidirectional movement of the DNA replication machinery
DNA Polymerase
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
releasing pyrophosphate
(PPi) + energy
DNA Polymerase
Bidirectional synthesis of the DNA double
 Corrects mistaken base pairings
 Requires an established polymer (small
RNA primer) before addition of more
 Other proteins and enzymes necessary
How is DNA Synthesized?
Original theory
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
Correction: Refer to
Figure 6-15 on page 205
of your textbook for
“corrected” figure. This
figure fails to show the
two terminal phosphate
groups attached on the 5’
end of the nucleotide
strand located at the top
of this figure.
How is DNA Synthesized?
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’
How is DNA Synthesized?
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
 Compensate
for this by feeding the DNA strand
through the polymerase, and primers and make
many short segments that are later joined (ligated)
Called the lagging strand
DNA Replication Fork Fig 6-12
Mistakes during Replication
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
Starting Synthesis
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)
Primers – cont’d
Simple addition of primer
along leading strand
RNA primer synthesized 5’
 3’, then polymerization
with DNA
Many primers are needed
along the lagging strand
1 primer per small
fragment of new DNA
made along the lagging
Called Okazaki fragments
Removal of Primers
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
 Requires
Mg2+ and ATP
Other Necessary Proteins
Helicase opens double helix and helps it
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
Components of the DNA Replication
Polymerase & Proteins Coordinated
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
 If no repair
In germ (sex) cells  inherited diseases
 In somatic (regular) cells  cancer
Effect of Mutation
Uncorrected Replication Errors
Mismatch repair
Enzyme complex recognizes mistake and excises
newly-synthesized strand and fills in the correct
Mismatch Repair – cont’d
Eukaryotes “label”
the daughter strand
with nicks to
recognize the new
Separates new from
Depurination or Deamination
Depurination – removal of a purine base from
the DNA strand
Deamination is the removal of an amine group
on Cytosine to yield Uracil
Could lead to the insertion of Adenine rather than
Guanosine on next round
Chemical Modifications
Thymine Dimers
Caused by exposure to UV light
2 adjacent thymine residues become
covalently linked
Different enzymes
recognize, excise
different mistakes
DNA polymerase
synthesizes proper
DNA ligase joins new
fragment with the