Dnareplication

DNA REPLICATION

Overview

A) CHROMOSOME STRUCTUREB) SEMICONSERVATIVE REPLICATIONC) THE REPLICATION PROCESSD) THE DNA BLUEPRINTE) THE GENETIC CODE

Chromosomes in Eukaryotic cells consist of:

DNAproteinsome

chromosomal RNA

Chromosomes in Prokaryotic cells consist of DNA only :

and so should not be called ‘chromosomes’

DNA has:

2. positively charged (basic) proteins called histones bonded to it

1. negative charges distributed along its length

Chromatin is the :

combination of DNA & histones

Functions of the histones:

1. organise 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) SEMICONSERVATIVE REPLICATIONC) THE REPLICATION PROCESSD) THE DNA BLUEPRINTE) THE GENETIC CODE

1. Semiconservative replication2. Conservative replication3. Dispersive replication

Three possible replication patterns:

Semiconservative replication

Conservative replication

Dispersive replication

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)

Three rounds of

replication:

Original DNA

1st Round:

2nd Round:

3rd Round:

0 min

20 min

40 min

60 min?

Overview

A) CHROMOSOME STRUCTUREB) SEMICONSERVATIVE REPLICATIONC) THE REPLICATION PROCESSD) THE DNA BLUEPRINTE) THE GENETIC CODE

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. A DNA polymerase III enzyme brings substrates to the template and catalyses 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!

DNA Template & dATP

New strand Template strand

5’ end 3’ end

Sugar A T

BaseC

G

G

C

A

C

TP

PP

OH

P P

3’ end

5’ end 5’ end

A T

C

G

G

C

A

C

T

3’ endPyrophosphate

2 P

OH

Phosphate

5’ end

deoxyribonucleoside triphosphate

nucleotide

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

DNA replication occurs in two steps:

2. The new nucleotides are linked by covalent bonding to each growing strand in a sequence determined by complementary base pairing.

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

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

Origin of replication

Site where DNA synthesis starts

A eukaryotic chromosome May have hundreds or even thousands of

replication origins

DNA is replicated simultaneously at the origins.

Replication fork is the :point at which the two strands of DNA are

separated to allow replication of each strand

• Each bacterial DNA has only one

Origin of replication

Directionality of the DNA strands at a replication fork

Leading strand

Lagging strand

Fork movement

Directionality of the DNA strands at a replication fork

Leading strand

Lagging strand

Fork movement

Protein RoleDNA helicases Unwinds the double helixRNA primase Synthesises RNA primersSingle-strand binding proteins

Keep the two strands separated

DNA polymerase I Erases primer and fills gapsDNA polymerase II [not in syllabus]

Proofreading of DNA

DNA polymerase III Synthesises DNA; proofreadingDNA ligase Joins the ends of DNA segments;

DNA repair

Replication: 1st step

• Unwind DNA– helicase enzyme• unwinds part of DNA helix• stabilised 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.

Then DNA polymerase III begins adding nucleotides to the 3 ′end of the primer.

Many Proteins at the Replication Fork

Identical base sequences

5’

5’

3’

3’ 5’

5’3’

3’

• DNA polymerases:1. can synthesise DNA only in the 5’ to 3’

direction 2. cannot initiate DNA synthesis

Problem at 3’ ends of Eukaryotic Chromosomes

Label structures at the Replication Fork

a. Leading strand templateb. Leading strandc. Lagging strandd. Lagging strand templatee. RNA primerf. Okazaki fragment

The Two New Strands Form in Different

Ways

Leading strand(continuous)

Lagging strand(discontinuous)

How are Okazaki fragments linked?

Each Okazaki fragment requires a

primer.

The final phosphodiester linkage between fragments is catalyzed by DNA ligase.

The Lagging Strand Story

The Lagging Strand Story

Many Proteins at the Replication Fork

Two dimensional view of a replication fork

Direction of synthesis

on lagging strand

Direction of synthesis on leading strand

3’5’

3’

5’3’5’

Proofreading procedure

• DNA replication is not perfect due to: 1) the high speed of replication

- (1000 nucleotides per second)2) spontaneous chemical flip-flops in the bases • occasionally DNA polymerase incorporates

incorrectly matched bases

If bases are paired incorrectly, the

nucleotide is removed.

Proofreading is done by several DNA polymerases including DNA polymerase II

Editing & proofreading DNA

• 1000 bases/second = lots of errors!

• 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

What is the advantage of the one-way directionality of the DNA structure?

Allows the proofreading enzymes to recognise the parental strand, running in one direction, as the ‘right stuff’.

Overview

A) CHROMOSOME STRUCTUREB) SEMICONSERVATIVE REPLICATIONC) THE REPLICATION PROCESSD) THE DNA BLUEPRINTE) THE GENETIC CODE

BLUEPRINT: a design plan or other technical drawing

DNA ‘Blueprint’

• every cell in the body has the same "blueprint" or the same DNA

• blueprint of a house tell the builders how to construct

a house

Importance of the DNA ‘Blueprint’

Tells the cell how to build

the organism.

How is it possible for cells to have:

the SAME DNA different structures & functions?

BUT

Proteins are a cell’s “molecular workers”

ANSWER:Every cell contains a particular set of proteins

Ovum must have receptors to bind the

sperm head.

Phagocyte must have receptors to engulf the microbe.

If all body cells have the SAME DNA, explain why only the pancreas makes insulin?

A cell has the ability to turn off most genes and only work with the genes

necessary to do a job.

DNA ‘Blueprint’

• information by itself, does not do anything – e.g. a blueprint may describe the structure of a house in great detail, but unless that information is translated into action, no house will ever be built

• likewise, although the base sequence of DNA, the “molecular blueprint” of every cell contains an incredible amount of information, DNA cannot carry out any action on it own

Central dogma: flow of information is from the:

DNA of a cell’s genes

the proteins that actually carry out the cell’s

functions

RNADNAProtein

to

What is ‘junk DNA’?

• 98.5% of human DNA does not code for proteins • Introns (old name: junk DNA) –

- the regions of DNA that do not code for proteins

• Exons –- the sections of DNA that code for proteins

Split genes:

• contain exons and introns • are found only in eukaryotic cells

Exons & Introns:

Gene

DNA

TranslationProtein A Protein B

Alternative splicing

Evidence for the role of DNA in inheritance: the

Hershey and Chase experiment (1952)

Martha ChaseAlfred Hershey

Hershey and Chase set out to determine whether the:

protein or DNA enters the bacterial cells.

• Bacteriophage - a particular type of virus which specifically attacks bacterial cells

• bacteriophage T2 :

attacks the bacterium Escherichia coli

consists of a protein coat and DNA

Which elements to follow?

DNA: in nucleotide

Protein:

BOTH proteins & DNA: C, H, O, N

S

P

in methionine + cysteine

This experiment confirmed that:

DNA from bacteriophages infected bacteria

Phagehead

Tail

Tail fiber

DNA

Bacterialcell

100

nm

DNA enters bacteria !!

OverviewA) CHROMOSOME STRUCTUREB) SEMICONSERVATIVE REPLICATIONC) THE REPLICATION PROCESSD) THE DNA BLUEPRINTE) THE GENETIC CODE

What does DNA code for?

DNA specifies only the production of protein synthesis

DNA nucleotide base sequence:

determines the amino

acid sequence of protein molecules

GENETIC CODE is the relationship between the: bases and amino acids

The code• DNA nucleotide bases:-

adenine, guanine, cytosine and thymine

• RNA has four nucleotide bases:- adenine, guanine, cytosine and uracil

• this ‘alphabet’ of 4 letters is responsible for carrying the code that results in the synthesis of a potentially infinite number of protein molecules

How many bases code for one amino acid? Recall that there are 20 different amino acids in proteins.

Only 4 amino acids would be possible. A, T, C, G1?

2?

3?

16 amino acids would be possible: still not large enough. e.g. AU, CU, or CC.

42 = 16

64 amino acids would be possible: e.g. AUU, GCG, or UGC. This vocabulary provides more

than enough words to describe the amino acids. 43 = 64

Conclusion:

The code is a triplet code i.e. three bases code for one amino acid.

Codon:a set of three adjacent nucleotides, also called triplet, in DNA or mRNA

that designates a specific amino acid to be

incorporated into a polypeptide

Six features of the genetic code

1. Triplet code2. Specificity3. Degeneracy4. Universality5. Non-overlapping6. Punctuated

1) The code is a triplet code• the DNA code for a protein is first copied into

messenger RNA (mRNA) before a protein is made

• mRNA is complementary to the DNA

DNA

mRNA

RNA base sequence

DNA

RNA

DNAA – TC – G

RNAA – UC – G

One mRNA molecule may contain hundreds or even thousands of bases

the cell recognises where the code for a protein starts and stops as the mRNA has:

START CODON

STOP CODON

start and

stop codons

64 codons in all

61 for amino acids

3 ‘stop codons’ (UAA, UAG, UGA)

1 ‘start codon’ (AUG – codes for methionine)

Codons in RNA

Methionine is specified by the codon AUG - known as the start codon

Note: it may be removed after the protein is synthesised

All proteins originally begin with the amino acid methionine. Why?

When the ribosome encounters a stop codon, it releases the :

1. newly synthesised protein

2. mRNA

2) The code is specific (non ambiguous)

• each triplet code specifies only one amino acid

• e.g. UUU = phenylalanine

3) The code is degenerate

ValineGUUGUCGUAGUG

a given amino acid may be coded for by more than one codon

64 codons and only 20 amino acids:

so some amino acids are coded for by several codons –

exceptions [next slide]:

TyrosineUAUUAC

LysineAAAAAG

TryptophanUGG

MethionineAUG

First TWO bases determine the amino acid

• Third Base is usually less specific than the first two.

• This is also known as the "Wobble Hypothesis" because often the:

ValineGUUGUCGUAGUG

third base can changeBUT

the amino acid remains the same.

Wobble position of a codon refers to the 3rd nucleotide in a codon

What is the advantage of a degenerate code?

This allows for possible mutations to be less damaging.

No change in polypeptide:

Polypeptide structure is changed

• deletion or addition of one or two bases, leads to a change in reading frame (reading sequence)

THE FAT CAT ATE THE BIG RAT

Delete C: THE FAT ATA TET HEB IGR AT

Insert A: THE FAT ATA ATE THE BIG RAT

Six features of the genetic code

1. Triplet code2. Specificity3. Degeneracy4. Universality5. Non-overlapping6. Punctuated

4) The code is nearly universal• the genetic code is the same in all organisms,

except in:

e.g. AGA = arginine in:all organisms whose genetic code has been studied

mitochondria protozoan nuclear DNAand

The universality of the genetic code is among the strongest evidence that all living things share a

common evolutionary heritage

What is the importance of the universality of the code?

GENETIC ENGINEERING IS POSSIBLE

Aim:to map out the entire genetic code of a human -2.1 million base pairs -(30,000 – 40,000 protein coding genes)

The Human Genome Project (1990 – 2003)

The Human Genome Project (1990 – 2003)

What is the ‘Genome’?

The total DNA in an organism

The human genome = 46 chromosomes

The total DNA in an organism

What is the size of a gene?

• average gene in humans: 3000 bases• but sizes vary greatly• the largest known human gene:

- 2.4 million bases

Six features of the genetic code

1. Triplet code2. Specificity3. Degeneracy4. Universality5. Non-overlapping6. Punctuated

5) The code is non-overlapping

non-overlapping:- no base of a given triplet contributes to part

of the code of the adjacent triplet

non-overlappingoverlapping

• the genetic code is read in groups (or “words”) of three nucleotides

• after reading one triplet, the “reading frame” shifts over the next three letters, not just one or two

Six features of the genetic code

1. Triplet code2. Specificity3. Degeneracy4. Universality5. Non-overlapping6. Punctuated

6. The code is punctuated:

REMEMBER: Excluding the start & stop codons, the actual code determining the sequence of amino acids is UNPUNCTUATED

NOTE: according to the syllabus, the code is punctuated due to start and stop codons

however

the majority of text books consider the code as being unpunctuated i.e. comma less

MUTATIONS

A mutation is a change in the

• amount, arrangement or structure of the DNA of an organism

A mutation produces a change in the genotype & is passed on when a cell nucleus divides by:mitosis or meiosis from the mutant cell

Mutant daughter cellsMutant daughter cells

Mutant cell

Mutant cell

Which type of mutation can be inherited by the offspring?

germinal

somatic Occur in somatic cells:are NOT passed on the offspring

Occur in gamete cells:are passed on to the offspring

A mutation may result in the change in appearance of a characteristic of a

population

e.g. red eyes in Drosophila appeared in 1909

e.g. dark-coloured moth appeared in 1848

The "typica" form of the moth.

The "carbonaria" form.

occur in: any gene at any timebe:

Mutations can

Spontaneous

Induced

Spontaneous Mutations: are permanent changes in the genome that

occur without any outside influence occur because the machinery of the cell is

imperfect

Both chromatids are sent to one

daughter cell, the other gets none.

One chromatid goes to each

daughter cell.

Induced Mutations: occur when some outside agent causes a

permanent change in DNA

mutagens: anything that causes a mutation examples:• Asbestos• Tar from tobacco• Ionising radiation e.g. UV• Pesticides• Caffeine

Mutation rates vary between organisms

In general, the mutation rate in:unicellular eukaryotes bacteria

Chernobyl disaster was a catastrophic nuclear accident that occurred on 26 April 1986

is roughly 0.003 mutations per genome per generation.

Chernobyl: mutant dog

Ionising radiation is radiation that: carries enough energy to liberate electrons from atoms or molecules, thereby ionizing them. 

Ionising radiation e.g. UV, X-rays, -rays

Ionising radiation damages the DNA

UV light causes adjacent thymines to

cross link

Mutations can be:

Chromosomal[covered in 2nd year]

Gene mutations or point mutations:

INSERTION

INVERSION

DELETION

SUBSTITUTION

describe a change in the structure of DNA at a single locus

1

2

Fig. 12 Gene or point mutation

1) INSERTION: the addition of an extra nucleotide

A GT G C A T A TT G A C A G

2) DELETION: involves the loss of a nucleotide

A GT G C A T A TT C A G

Fig. 12 Gene or point mutation

4) SUBSTITUTION: a particular base is substituted by another (e.g. sickle-cell anaemia)

A GT G C A T A TT G T A G

3) INVERSION: two nucleotides become arranged in the wrong order

A GT G C A T T TA G C A G

Sickle Cell Anaemia in humans is an example of base substitution

• a base in one of the genes involved in producing haemoglobin is substituted

• at position 14 in the DNA:thymine is replaced by adenine

Sickle Cell Anaemia: at low oxygen tensions, haemoglobin S

crystallises in the red cells distorting them into a sickle shape

Point mutations

No mutation

DNA level TTC TTT ATC TCCmRNA level

AAG AAA UAG AGG

Protein level

Lys Lys STOP Arg

Silent Nonsense Missense

Missense mutation

Nonsense mutation

is a point mutation in a sequence of DNA that results in a premature stop codon

is a point mutation that results in the substitution of one amino acid in protein for another

Frameshift mutationsThe addition or

deletion of a single base

has much more profound consequences than does the substitution of one base for another

THE CAT SAW THE DOG

A frameshift mutation:alters the reading frame in the mRNA

downstream of the mutation

TA deleted

Changing the reading frame early in a gene, and thus in its mRNA transcript, means that the

majority of the protein will be altered.

Amino acidDeletion of a single nucleotide

DNAbases

Original DNA code for an amino acid sequence.

Incorrect amino acid sequence, which may produce a malfunctioning protein.

End-Of-Year SEP 2013Use your knowledge of the genetic code to explain statements (a) and (b) below. Use your knowledge of genetic mutations to answer statements (c), (d) and (e). [5 marks each]

i) Distinguish between a base substitution and an inversion.

i) Distinguish between a deletion and an insertion.ii) Explain how deletions and insertions lead to

frameshift mutations

Use your knowledge of biology to explain the following.The structure of the DNA molecule permits vast amounts of information to be stored. (5 marks)

Question: [SEP, 2007]

1. Information on the DNA molecule is in the form of a sequence of bases, where three consecutive bases specify an amino acid. Thus a small number of bases are needed to code for an amino acid. Considering that DNA within a eukaryotic cell is 2m long, it allows for a large amount of information to be stored.

2. In many eukaryotic cells, split genes occur. These contain regions which code for the protein called exons and introns which do not code. The way in which the exons are linked together determines the type of polypeptide to be formed. Thus one gene can form a number of closely related polypeptides.

THE END