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

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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