Bacterial and Eukaryotic DNA
REPLICATION
Overview
A) CHROMOSOME STRUCTUREB) MODE OF REPLICATIONC) THE REPLICATION PROCESSD) CONFORMATIONS OF
DNA REPLICATION
Genetic material of Prokaryotic cells consist of DNA only :
and so should not be called ‘chromosomes’
Bacterial DNA Structure
• Bacteria have closed, circular DNA• Genome: genetic material in an organism• E. coli– 4 million base pairs– DNA- 107Ȧ long and 20 Ȧ in diameter– cell–2x103 Ȧ long and 8x104 Ȧ in diameter– DNA takes up around 10% of cell volume
Genetic material of Eukaryotic cells consist of:
DNAprotein
DNA has:
2. positively charged (basic) proteins called histones bonded to it
1. negative charges distributed along its length
Functions of the histones:
1. organize the chromosome physically2. regulate the activities of the DNA
histones
Nearly 2m of DNA are crammed into each human
cell.What does this mean?
A great deal of information can
be stored!!
A closer at DNA:
Four Key elements of DNA structure
1) a double-stranded helix
2) of uniform diameter
3) twisting to the right
4) the two strands running in opposite directions
Overview
A) CHROMOSOME STRUCTUREB) MODE OF REPLICATIONC) THE REPLICATION PROCESSD) CONFORMATIONS OF
DNA REPLICATION
1. Semiconservative replication2. Conservative replication3. Dispersive replication
Three possible replication patterns:
Semiconservative replication
Conservative replication
Dispersive replication
What do you think is the acceptable mode of replication today?
Semiconservative replication
Each parent strand serves as a template for a new strand and the two new DNA strands each
have one old and one new strand
Parent strands
New / daughter strand
Meselson and Stahl experiment [1958] demonstrates
semiconservative replication:
Cells broken open to extract DNA
E. coli grown in the presence of 15N (a heavy isotope of Nitrogen) for many generations
E. coli placed in medium containing
only 14N (a light isotope of Nitrogen)
• Cells get heavy-labeled DNA
Sampled
at:
0 min
1
2
3
40
min
20
min
Suspended DNA in cesium chloride (CsCl) solution.
4
15N medium
CsCl density gradient centrifugation
5
15N14N
DNA
Both strands heavy
F1 generation DNA (one heavy/one
light strand)
0 min 20 min 40 min
F2 generation DNA:
Two light strands
(one heavy/one light strand)
Overview
A) CHROMOSOME STRUCTUREB) SEMICONSERVATIVE REPLICATIONC) THE REPLICATION PROCESSD) CONFORMATIONS OF
DNA REPLICATION
DNA RNA protein
transcription translation replication
reverse transcription
Central dogma
22
What is the significance of DNA replication?
• For the transfer of genetic information with high fidelity
-from cell to cell -from one generation to another
FOUR requirements for DNA to replicate
1. DNA to act as a template for complementary base pairing.
2. The four deoxyribonucleoside triphosphates:
dATP, dGTP, dCTP & dTTP.
The nucleotides arrive as nucleosides– DNA bases with P–P–P• P-P-P = energy for bonding
– DNA bases arrive with their own energy source for bonding
dATP dGTP dTTP dCTP
3. A source of chemical energy is needed to drive this highly endergonic reaction.
DNAPolymerase III
4. Enzymes that catalyzes the reactions
energy
ATPGTPTTPCTP
Energy of ReplicationWhere does energy for bonding usually come from?
ADPAMPGMPTMPCMPmodified nucleotide
energy
We comewith our ownenergy!
And weleave behind anucleotide!
YourememberATP!Are there other waysto get energyout of it?
Are thereother energynucleotides?You bet!
Basic rules of DNA replication
• Specificity of base pairing
• Synthesis direction 5’-3’ (C at 3’ position in 3’ end has free –OH)
• Nucleotides added at 3’ of growing strand – DNA polymerase
Components of DNA Replication DNA polymerases- Deoxynucleotide polymerization
Helicase -Processive unwinding of DNA
Topoisomerases Relieve torsional strain that results from helicase-induced unwinding
RNA primase Initiates synthesis of RNA primers
Single-strand binding proteins Prevent premature reannealing of dsDNA
DNA ligase Seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand
DNA replication occurs in two steps:
1. DNA is locally denatured (unwound)
WHY?
To separate the two template strands and make them available
for base pairing.Unzipping of
DNA
What are the bonds present in DNA?
2. The new nucleotides are linked by covalent bonding to each growing strand in a sequence determined by complementary base pairing.
Bacterial DNA Replication
Enzymes
Three Stages of replication
1) Initiation – occurs at the origin of replication
2) Elongation
– involves the addition of new nucleotides based on complementarity of the template strand
3) Termination
– occurs at a specific termination site
• Each bacterial DNA has only one origin of replication
Initiation
• Unwind DNA– helicase enzyme• unwinds part of DNA helix• stabilized by single-stranded binding proteins
single-stranded binding proteins replication fork
helicase
A primer is :- required to start
DNA replication—a short single strand of RNA.
- synthesised by primase.
Many Proteins at the Replication Fork
Elongation Once priming is complete DNA pol III
is loaded into the DNA and replication begins
Nucleophilic attack by the 3' OH on the alpha phosphate releases pyrophosphate
Subsequently hydrolyzed (by inorganic phosphatase) into two phosphates
This hydrolysis drives DNA synthesis to completion
REMEMBER:
Nucleotides are always added to the growing strand at the 3’ end – the end at which the DNA strand has a free –OH group on the 3’ carbon of its terminal deoxyribose
Protein complexes of the replication fork
Bacterial DNA Polymerase
How are Okazaki fragments linked?
Each Okazaki fragment requires a
primer.
The final phosphodiester linkage between fragments is catalyzed by DNA ligase.
Termination
• In some DNA molecules, specific termination sequences block further replication. – A termination protein, called
Tus in E. coli, binds to these sequences. Tus blocks the movement of helicase, thus stalling the replication fork and preventing further DNA replication.
In some DNA molecules, replication is terminated whenever two replication forks meet.
Eukaryotic DNA Replication
• The eukaryotic origins are shorter than that of E. coli.
• Requires DNA-pol (primase activity) and DNA-pol (polymerase activity and helicase activity).
• Needs topoisomerase and replication factors (RF) to assist.
Initiation
52
• DNA replication and nucleosome assembling occur simultaneously.
• Overall replication speed is compatible with that of prokaryotes.
Elongation
53
DNA Polymerase in Eukayotes
3'
5'
5'
3'
3'
5'
5'
3'
connection of discontinuous
3'
5'
5'
3'
3'
5'
5'
3'
segment
Termination
55
A eukaryotic chromosome May have hundreds or even thousands of
replication origins
DNA is replicated simultaneously at the origins.
Proofreading procedure
• DNA replication is not perfect1) the high speed of replication
- (1000 nucleotides per second)2) spontaneous chemical flip-flops in the bases • occasionally DNA polymerase incorporates
incorrectly matched bases
Errors in Replication
For Bacterial cells -error is at a frequency of 1 in 109 to1010
nucleotides
For Eukaryotic cells -error is expected to be one every 115.7
days to 1157.4 days
• A much higher rate of error in replication is incorporated in
exposure to various mutagens (e.g. X-ray)
Editing & proofreading DNA
• 1000 bases/second
• DNA polymerase I – proofreads & corrects mistakes – repairs mismatched bases– removes abnormal bases
• repairs damage throughout life
– reduces error rate from 1 in 10,000 to 1 in 100 million bases
Fast & accurate!
• It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome – divide to form two identical daughter cells
• Human cell copies its 6 billion bases & divide into daughter cells in only few hours– remarkably accurate– only ~1 error per 100 million bases– ~30 errors per cell cycle
Overview
A) CHROMOSOME STRUCTUREB) MODE OF REPLICATIONC) THE REPLICATION PROCESSD) CONFORMATIONS OF
DNA REPLICATION
CONFORMATIONS OF DNA REPLICATION
• LINEAR DNA REPLICATION
• CIRCULAR DNA :THETA CONFORMATION
• CIRCULAR DNA :ROLLING CIRCLE CONFORMATION
Conformations of Replication
THE END