DNA replication

Lecture 3-1

Review BEFORE you come to lecture

1. Review molecule of DNA.

2. Compare and contrast DNA and RNA.

3. Read the genetic code on a DNA strand and draw the

complementary strand.

DNA strand A G G C T A A G C
Not in lecture

Complementary

strand

• View the videos posted on Canvas and write your notes here:

Not in lecture

After this lecture you should be able to (aka
Learning Objectives)

• State why Crick, Franklin, Watson, Wilkins and Franklin are important for

the discovery of DNA molecule, its structure and its function.

• Describe the molecular structure of DNA.

• Compare and contrast DNA and RNA.

• Describe how leading and lagging strands synthesize, and why lagging

strand lags.

• Read the genetic code on a DNA strand and draw the complementary

strand.
Not in lecture

• List proteins required for DNA replication and state their functions.

• Describe proofreading by DNA polymerase III, and repair by nucleotide

excision repair mechanism.

Appropriate terminology

Enzymes and other proteins

Our big

picture today

Process

5
www.abitur-wissen.org

What are our topics today?

1. A short history about discovery of DNA

2. Structure of DNA - a quick review (not in lecture). Please don’t get

scared off by the number of slides. These slides condense basics

about DNA, which you most likely already know.

3. DNA replication

The most difficult in this lecture is:

• Understanding how replication of the lagging strand is done (part of

topic 3)

A short history about the

discovery of DNA

Topic 1

Guess what!

• Before DNA was not considered genetic material, which

macromolecules were thought to be storing genetic

information? Why?

a. Lipids

b. Proteins

c. Carbohydrates

Friedrich Miescher

ice W ilkins was the the first to

Maur he Nobel

t publish about

shared 962 with nucleic acids in

n1
Prize i and Crick.

n 1871.

Watso

Photograph 51

James
Wa
Francis tson and

the firs rick were

t to pu
about b

DNA st lish
ru

in 1953 cture

Rosalind Franklin
Dieearlydueto
10

radioactive
materiel
(1920-1958)


Hint: these kinds of

Let’s stop and review questions may appear

on your tests

• Given a polynucleotide sequence such as GTTC, can you tell which is

the 5’ end? If not, what further information do you need to identify

the ends?

Not in lecture

which has an –OH group on the 3’ carbon (the 3’ end) (check figure 14.3 in your set of slides).

ANSWER: You can’t tell which end is the 5’ end. You need to know which end has a phosphate group on the 5’ carbon (the 5’ end) or
11

Structure of DNA – a quick

review

Topic 2

Not in lecture

12

What is DNA and its structure? Figure 4.1

• DNA is a nucleic acid

• DNA = deoxyribonucleic acid

• Made of long chains (polymers of

nucleotides)

• Each nucleotide consists of a:

• Phosphate group
Not in lecture

• Deoxyribose sugar group

• Nitrogen-containing base

• Observe the figure, and state how

nucleotide and nucleoside differ.

13

DNA has four kinds of bases

• Pyrimidines are thymine

and cytosine, and have

one ring.
• Purines are adenine, and

Not in lecture

guanine, and they have

two rings.

Figure 4.1

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DNA’s primary structure

• Nucleotides are

monomers that form

the DNA polymer.

• DNA’s primary

structure is made up
Not in lecture

of a sequence of

nucleotides.

Figure 15.3

15

Recall the directionality of

DNA’s primary structure DNA strands:

Note two free ends:

5’ end (phosphate attached to a 5’

carbon) and

3’ end (hydroxyl group on a 3’ carbon)

Note how adjacent nucleotides are

joined vertically by covalent bonds

called phosphodiester bonds.
Not in lecture

Directionality of DNA molecule is from 5’ to 3

Figure 15.3 16

Complementary base pairing

Thymine and adenine form 2 hydrogen bonds

Not in lecture

Guanine and cytosine form 3 hydrogen bonds

Figure 4.6

17

DNA’s secondary structure

DNA molecule is a double-stranded helix, each strand

with a sugar-phosphate backbone.

Each of the strands has nitrogenous bases projecting

and forming hydrogen bonds between the 2 strands.

Why hydrogen bonds? They are strong enough to hold bases
Not in lecture

together, but also weak enough to break when required.

These 2 long strands run in opposite 5’->3’

directions, which is termed antiparallel.

Figure 15.4
18

A quick hint on how to the tell where the 5’
end is

Technically speaking,

the double-helix is

composed of 2

polynucleotide

molecules held

together by hydrogen

bonds.

The oxygen faces

Not in lecture

The oxygen faces towards the 5’

towards the 5’ end

end

Source: Campbell Biology 19

DNA replication – a quick reminder

• The parent strand acts as a template for DNA replication

• Joining base pairs: A and T, G and C (energetically the most favourable of all

possibilities)

• This is known as semiconservative replication = each newly made DNA molecule

comprises one old strand and one new strand.

T

A T A A T A T A T

C G C G G C G C G
C

G C G C C G C G C
Not in lecture

A
A T A T A T A T

Free
T A T A T A T A

nucleotides

A parental The parental strands Two identical

molecule separate and serve daughter molecules

of DNA as templates of DNA are formed
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DNA replication

Topic 3

21

I passed
away at 88

DNA replication I, at

89

• What is DNA replication?

• A duplication of DNA during cell

reproduction.

• Why does it happen?

• The chromosomes need to

duplicate so that the daughter

cells can end up with same • Who proposed the DNA

amounts of DNA. replication model?

• Watson and Crick proposed

• When does it happen? both, the DNA structure and

• During S phase of the interphase. its replication model.

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How does DNA replication proceed?

NOTE 3: We will mostly be moving back and forth

NOTE 1: the enzyme called DNA polymerase is

from bacterial to eukaryotic models, for

crucial for the DNA replication process. There

simplification.

are several types of DNA polymerase enzymes

in organisms, but we will consider the following

Step 1. The
two: polymerase I, and polymerase III, which

origin of are used during DNA replication.

replication Step 2. New

Hasto be
DNA strand

in I IIItIv

elongation Step 3. Anti-

parallel

NOTE 2: DNA polymerase can add new

elongation Step 4.

nucleotides only to the 3’ end of the growing

DNA chain. Hence the DNA elongation always Proofreading

proceeds in the 5’->3’ direction. and repair

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Old DNA is a template; new DNA

Step 1. The origin of replication

is newly-synthesized DNA.

Replication fork is the place where

The origin of replication is a stretch of new DNA strand elongates and is

DNA, which forms a “bubble”. catalyzed by DNA polymerase.

sizeofthe
The DNA
Why?

Thecircularshape

oftheDNA

Bacteria have a single

point of origin, while

eukaryotes have many.

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Figure 15.7

Step 2. Elongation of the new DNA

DNA polymerase III is required

Figure 15.6

bad new nucleotide

As each monomer joins

the growing end of the

DNA strand, it looses 2 P

groups. Subsequent

hydrolysis of the

diphosphate (P-P), to 2

molecules of P (inorganic)

is the exergonic reaction

yB'can
elongation
that drives the elongation

Nucleoside triphosphate helpof of the DNA polymer.

withthe

with high potential energy polymerase

ever source 25

Previous figure in words

• When adding new nucleotides onto an elongating DNA molecule, note that

nucleotides do not float freely in the cytoplasm. Instead, it is the nucleoside

triphosphates (each composed of a sugar and a base with 3 phosphate groups)

that float in cells.

• Adding new nucleotides can only occur at the 3’ end, because at the 5’ end the

strand already carries a phosphate group. This reaction is exergonic in the cells

(and not endergonic as you learned about polymerization reactions in chemistry),

because nucleoside triphosphates have 3P groups that have high potential
energy.
Not in lecture

• What adds these monomers? The enzyme DNA polymerase does.

• To summarize: individual nucleotides are added as a part of a DNA elongation

process. Enzyme DNA polymerase is needed for this elongation, as well as energy

obtained from breaking off 2 phosphate molecules from the nucleosides.

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Step 3. New DNA leading strand anti-parallel

elongation (step 3-1)

bytwisting
closingtheDNA

enzymetwistopenDNA

Not an enzyme!

Notan
Not
Not anenzyme!
an enzyme!
enzyme!

27

Figure 15.8


Step 3. New DNA leading strand anti-parallel

elongation (step 3-2)

Not an enzyme!

Not an enzyme!

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Figure 15.8


Previous figure in words

• DNA polymerase III requires a primer. What is a primer? It is always an

RNA stretch, complementary bonded, and providing a free 3’ OH group.

• Why is there a need for the primer? Because the primer initiates the DNA

synthesis – DNA polymerase III cannot add additional nucleosides if there is

no primer present.

• Why is it often an RNA primer and not a DNA one? RNA polymerase can

synthesize RNA from scratch, while DNA polymerase cannot. Primer
provides a free 3’-OH group that can combine with the incoming
Not in lecture

nucleoside triphosphates to form a phosphodiester bond.

• Note the 5’-3’ direction – this is the direction of the newly synthesized DNA

strand! This is important to remember.

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Now you do…

primase
1. Which enzyme is this?_____________

2. Why are these

proteins here?

To

stabilize single

strands.

3. What does a primer provide?

a free 3’-OH group that can combine with the incoming nucleoside
Primer provides _____________________

triphosphates to form a phosphodiester bond.

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Step 3. New DNA lagging strand anti-parallel

elongation (step 3-1)

Direction of new DNA synthesized using

leading strand as a template (INTO the fork)

“old” DNA

“old” DNA

Direction of new DNA synthesized

using lagging strand as a template

(AWAY from the fork)

Figure 15.10

31

Previous figure in words

• Note that the synthesis of the leading and lagging strands occurs

concurrently (at the same time) and at the same rate (same speed). The

lagging strand is so named because its synthesis is slightly delayed relative

to synthesis of the leading strand.

• Top strand of the “old” DNA is 3’, bottom is 5’; new strand goes 5’->3’

direction.

• The reasons why the lagging strand lags: DNA is a double helix, with 2

antiparallel strands that run in opposite directions. In addition, DNA
polymerase III can add nucleotides only to the 3’ end. Therefore, DNA
Not in lecture

polymerase III that is synthesizing the leading strand works INTO the

replication fork, but a second DNA polymerase III that is working on the

lagging strand, must do so AWAY from the the fork, in order to keep the

other strand in 5’->3’ direction.

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Step 3. New DNA lagging strand anti-parallel

elongation (step 3-2)

Figure 15.10

33

Previous figure in words

• Once primase synthesizes a short RNA primer on the lagging strand,

DNA polymerase III synthesizes short fragments of DNA along the

lagging strand. These short fragments, containing a primer and a

short stretch of newly-synthesized DNA, are called Okazaki fragments

and the first such fragment is shown in this figure. It is important to

be able to explain what Okazaki fragments are and where we find

them.
Not in lecture

• Later, these fragments seal together forming a continuous whole.

• Okazaki fragments are not of the same length among organisms. For

example, they are ~1,000 – 2,000 nucleotides long in E. coli, and

~100-200 in eukaryotes.

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Step 3. New DNA lagging strand anti-parallel
elongation (step 3-3)

primers

15.10
Figure
35

Previous figure in words

• Here comes the 2nd Okazaki fragment!

• Note that each Okazaki fragment requires a primer, so the lagging

strand will have a new primer for each of the Okazaki fragments,

while the leading strand will have only one primer for the whole

strand – this is important to distinguish.

Not in lecture

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Step 3. New DNA lagging strand anti-parallel

elongation (step 3-4)

Figure 15.10

Each primer = RNA stretch

is now replaced with DNA

nucleotides by DNA polymerase I 37

Step 3. New DNA lagging strand anti-parallel

elongation (step 3-5)

A gap between 2 Okazaki fragments

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Figure 14.10

SUMMARY TABLE 15.1

Pay attention to this table

Table 15.1 is here as a reminder to go

over it, since it clearly summarizes

proteins required for DNA synthesis

and their function. Know them all.

Not in lecture

Note: DNA polymerase I is missing on the list of

proteins and enzymes for leading-strand synthesis

39

Why do errors in DNA synthesis occur and

what fixes them?

Usually fixed by DNA polymerase III itself

What are the reasons for errors in DNA

synthesis?

• Incorrectly paired (mismatched)

nucleotides that escape DNA

polymerase proofreading

• DNA molecules are subject to

constant harmful chemical and

physical agents (cigarette smoke, X https://theosophical.files.wordpress.com/2014/12/telephone-game.jpg

rays, UV, etc.)

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Step 4. Proofreading and repair (by DNA polymerase)

Note that during such repair,

DNA polymerase replaces

one nucleotide at the time.

DNA pol III

Figure 15.15

DNA polymerase III can

proofread!
A
DNA pol III
41
 Why do errors in DNA synthesis occur and
what fixes them?
Usually fixed by DNA polymerase III itself
What are the reasons for errors in DNA
synthesis?
• Incorrectly paired (mismatched)
nucleotides that escape DNA
polymerase proofreading
• DNA molecules are subject to
constant harmful chemical and
physical agents (cigarette smoke, X https://theosophical.files.wordpress.com/2014/12/telephone-game.jpg

rays, UV, etc.)

Usually fixed by nucleotide excision repair systems 42
 Step 4. Proofreading and
repair (by nucleotide excision
repair system)
Damaged DNA (a covalent bond between 2 T,
causes the DNA to buckle and interfere with
DNA replication). This is often caused by UV.

Cutting the damaged DNA by an enzyme (called

nuclease) with “molecular scissors” feature

DNA polymerase fills in the missing nucleotides.

DNA ligase seals DNA pieces.

Figure 15.17
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Summary
• What the origin of replication means
• How DNA polymerase and other enzymes work to elongate
the new DNA in 5’-3’ direction
• How the elongation of the new DNA happens in antiparallel
manner
• How mistakes may happen, and how the cell corrects them

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Key concepts
• DNA is the genetic material.
• A chromosome consists of a DNA molecule packed together
with proteins.
• Many proteins work together in DNA replication and repair.

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 Core concepts
• In DNA replication, a single parental molecule of DNA produces two
daughter molecules.
• The replication of linear chromosomal DNA requires mechanisms that
ensure efficient and complete replication.
Not in lecture

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 Related videos
• On Canvas:
• DNA replication.m4v
• Nucleotide excision repair
Not in lecture

47
 1. What was the most important
thing you’ve learned in class today?

2. What questions do you have?

Exit slip questions
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 From gene to protein
Next time
Not in lecture

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