Chapter 5 & 6 - DNA & DNA Replication

Chapter 5 & 6
DNA & DNA Replication
History

DNA
Comprised of genes
 In non-dividing cell nucleus
as chromatin

 Protein/DNA

complex
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

Nucleotides




A, G, T, C
Sugar and phosphate
form the backbone
Bases lie between the
backbone
Held together by
H-bonds between the
bases


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
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

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
begins



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
helix
 Corrects mistaken base pairings
 Requires an established polymer (small
RNA primer) before addition of more
nucleotides
 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.
Why DNA
Isn’t
Synthesized
3’5’
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
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
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
Proofreading
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
strand
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
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)
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
assault
 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
pairing
Mismatch Repair – cont’d

Eukaryotes “label”
the daughter strand
with nicks to
recognize the new
strand

Separates new from
old
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
Repair
Mechanisms



Different enzymes
recognize, excise
different mistakes
DNA polymerase
synthesizes proper
strand
DNA ligase joins new
fragment with the
polymer