27/11/17

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

DNA REPLICATION,
REPAIR AND
RECOMBINATION
Subject 012

27/11/17 1

Jurusan
Kimia
Topic   Learning  outcomes   Dr.  rer.  nat.  Syafrizayanti  

1. Overview of DNA •  Understand that DNA polymerase requires a template and primers to synthesize DNA.
Replication   •  Understand that double-stranded DNA is replicated semidiscontinuously.  

2. Prokaryotic DNA •  Understand that DNA polymerases include 3ʹ → 5ʹ exonuclease activity and, in the case of
Replication   Pol I, 5ʹ → 3ʹ exonuclease activity.
•  Understand that DNA replication requires additional proteins to separate DNA strands, a
sliding clamp is loaded onto the template DNA to enhance the processivity of DNA
polymerase, DNA ligase, a termination factor, and topoisomerases.
•  Understand that balanced nucleotide levels, the polymerase mechanism, its proofreading
mechanism, and the activity of DNA repair enzymes contribute to the accuracy of DNA
replication.  
3. Eukaryotic DNA Replication   •  Understand that eukaryotic DNA replication requires DNA polymerases with different
degrees of accuracy and processivity.
•  Understand that pre-replication complexes assemble at multiple origins distributed
throughout the eukaryotic genome.
•  Understand that telomerase uses an RNA template to extend the 3_ ends of eukaryotic
chromosomes.  
4. DNA Damage   •  Understand that DNA is susceptible to damage from a variety of sources.
•  Understand that mutagenicity, which is related to carcinogenicity, can be tested.  
5. DNA Repair   •  Understand that some DNA damage can be repaired by the action of a single enzyme.

6. Recombination   •  Understand that in homologous recombination, DNA strands cross over to exchange
places.
•  Understand that recombination can repair damaged replication forks and double-strand
breaks.
•  Understand that transposons move themselves and sometimes additional genes to new
chromosomal locations.  
27/11/17 2

1  
 27/11/17  

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 3

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 4

2  
 27/11/17  

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 5

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 6

3  
 27/11/17  

Jurusan
DNA Kimia
•  DNA is an extremely storageDr.  medium
rer.  nat.  Syafrizayanti  

(cell infromation)
•  This does not mean it is static
(unchanging)
•  Somewhere in the DNA molecule, DNA
is constantly being replicated, repaired,
or transcribed into RNA
•  Complex protein machines are required
to maintain the stability of DNA’s
information

en.wikipedia.org
27/11/17 7

Prokaryotes genes are lowercase

J u r uinsitalic
an
Kimia
Dr.  rer.  nat.  Syafrizayanti  

Genes products
(proteins) are capitalized
normal type
27/11/17 8

4  
 27/11/17  

Jurusan
DNA replication models Kimia
Dr.  rer.  nat.  Syafrizayanti  

mun.ca/biology/scarr/iGen3_03-0

27/11/17 9

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 10

5  
 27/11/17  

Jurusan
Overview of DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

Learning •  Understand that DNA polymerase requires a template

Objectives   and primers to synthesize DNA.
•  Understand that double-stranded DNA is replicated
semidiscontinuously.  

27/11/17 11

Jurusan
Overview of DNA replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 12

6  
 27/11/17  

Jurusan
Overview of DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  DNA is replicated by enzymes known as

DNA polymerases
•  DNA polymerase requires single-stranded
DNA (ssDNA) as template and primers to
synthesize new DNA.
•  DNA replication occurs at replication forks.
•  Double-stranded DNA is replicated
semidiscontinuously

27/11/17 13

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 14

7  
 27/11/17  

Semidiscontinuous DNA replication Jurusan

Kimia
“Two complementary strands are synthesized at on once”
Dr.  rer.  nat.  Syafrizayanti  

•  Both daughter strands (leading strand

red, lagging strand blue) are synthesized
in their 5’ → 3’ direction.

•  The leading strand is synthesized

continuously, whereas the lagging strand
is synthesized discontinuously.

•  The lagging strand can only be made

discontinuously segments are known as
Okazaki fragments
•  Okazaki fragments are 100–200 nt long,
found by Reiji Okazaki, 1968

27/11/17 15

Jurusan
DNA Polymerase Catalytic Mechanism Kimia
Dr.  rer.  nat.  Syafrizayanti  

The reaction occurs through the

nucleophilic attack of the
growing DNA chain’s 3’ -OH group on
the phosphoryl of an incoming
nucleoside triphosphate.

A DNA polymerase requires two

metal ions

The incoming nucleotides are

selected by their ability to form
Watson–Crick base pairs with the
template DNA

27/11/17 16

8  
 27/11/17  

Jurusan
The elongation reaction Kimia
Dr.  rer.  nat.  Syafrizayanti  

q  Breaking one phosphate bond (in the NTP) but

forming another (in the growing strand)
q  However, base-stacking interactions with the
base behind it and base-pairing hydrogen
bonds with the base across from it stabilze the
new base and push the reaction forward
q  Also, PPi is hydrolyzed by inorganic phasphates
q  End result, thermodynamically favored
reaction at the cost of an NTP molecule

27/11/17 17

Jurusan
Two primary types of enzymes process DNA Kimia
Dr.  rer.  nat.  Syafrizayanti  

q  The one you will see in action was DNA polymerase,
an enzyme that can create DNA

•  E. coli has four distinct polymerase

•  Eukaryotes have different polymerases as well

q  Enzymes that cut DNA or break it apart are called

nucleases

•  Exonucleases chew on DNA ends

•  Endonucleases can attack in the middle of strand

27/11/17 18

9  
 27/11/17  

Jurusan
Requirements for elongation Kimia
Dr.  rer.  nat.  Syafrizayanti  

1)  A polymerase
2)  NTPs to add onto the growing strand
3)  A template to guide the process
•  An opposite strand to tell the polymerase how
to order the nucleotides
4)  A primer to start the growing strand
•  Polymerase elongates, it does not begin , it
needs a short complementary strand to start
adding on to

27/11/17 19

Jurusan
Prokaryotic DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

Learning Objectives   •  Understand that DNA polymerases include 3ʹ → 5ʹ

exonuclease activity and, in the case of Pol I, 5ʹ → 3ʹ
exonuclease activity.

•  Understand that DNA replication requires additional

proteins to separate DNA strands, a sliding clamp is
loaded onto the template DNA to enhance the processivity
of DNA polymerase, DNA ligase, a termination factor, and
topoisomerases.

•  Understand that balanced nucleotide levels, the

polymerase mechanism, its proofreading mechanism, and
the activity of DNA repair enzymes contribute to the
accuracy of DNA replication.  

27/11/17 20

10  
 27/11/17  

Jurusan
Replication of circular chromosome Kimia
Dr.  rer.  nat.  Syafrizayanti  

E. coli chromosome is a circular supercoiled DNA molecule of 4.6 106 bp.

•  Replication starts at an
origin (oriC), and is
bidirectional producing a
“bubble”θ
•  DnaA bound to oriC recruits
two hexameric complexes of
DnaB (Helicase)
•  Single-strand binding
protein (SSB) prevents
DNA from reannealing

27/11/17 21

Prokaryotic DNA Replication: Enzymes and Their J u r u s a n

Kimia
Functions Dr.  rer.  nat.  Syafrizayanti  

Ezyme/protein Specific Function

DNA Pol I* Exonuclease activity removes RNA primer and replaces with
newly synthesized DNA
DNA Pol II Repair function
DNA Pol III Main enzyme that adds nucleotides in the 5’à 3’ direction
Helicase Opens the DNA helix by the breaking hydrogen bonds
between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments to create
one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Sliding clamp Helps to holds the DNA polymerase in place when
nucleotides are being added
Topoisomerase Helps relieve the stress on DNA when unwinding by causing
breaks and resealing the DNA
Single strand binding Binds to sngle stranded DNA to avoid DNA rewinding back
proteins (SSB)

27/11/17 22

11  
 27/11/17  

Comparison of Three DNA Polymerase of Jurusan

Kimia
E.coli Dr.  rer.  nat.  Syafrizayanti  

27/11/17 23

Jurusan
Overview Prokaryotic DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  DNA polymerases include 3ʹ → 5ʹ exonuclease activity and, in

the case of Pol I, 5ʹ → 3ʹ exonuclease activity.
•  DNA replication requires additional proteins to separate DNA
strands, unwind the helix, bind to single-stranded DNA, and
synthesize RNA primers.
•  A sliding clamp is loaded onto the template DNA to enhance
the processivity of DNA polymerase.
•  Complete replication of DNA requires DNA ligase, a
termination factor, and topoisomerases.
•  The balanced nucleotide levels, the polymerase mechanism,
its proofreading mechanism, and the activity of DNA repair
enzymes contribute to the accuracy of DNA replication.
27/11/17 24

12  
 27/11/17  

Polymerase I has Jurusan

Kimia
Exonuclease Activity Dr.  rer.  nat.  Syafrizayanti  

27/11/17 25

The Leading and Lagging Strands Are Synthesized Jurusan

Simultaneously Kimia
Dr.  rer.  nat.  Syafrizayanti  

The lagging strand template must loop around to permit the holoenzyme to extend the primed lagging strand

27/11/17 26

13  
 27/11/17  

The replication of E. coliJ uDNA.

rusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  Replication initiation requires

Helicase to unwind DNA
Primosome •  Single strand binding protein
(SSB) prevents DNA from
reannealing
•  The primosome synthesizes
RNA primers.

27/11/17 27

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  A sliding clamp (β-clamp)

promotes Pol III
processivity
•  β-clamp structure
determined by John
Kuriyan
•  It is a dimer of C-shaped
monomers that form an
80-Å diameter donut-
shaped structure

27/11/17 28

14  
 27/11/17  

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  
The lagging strand loops back around to the
same DNA polymerase III molecule

27/11/17 29

The replacement of RNA primers by DNA in Jurusan

Kimia
lagging strand synthesis Dr.  rer.  nat.  Syafrizayanti  

1)  The RNA primer (Fragment 1) is

excised by Pol I and then the
enzyme add dNTPs to the 3 end
of the newly synthesized Okazaki
fragment (Fragment 2)

2) The nick is sealed by the action

of DNA ligase

27/11/17 30

15  
 27/11/17  

Nick translation can be useful to prepare Jurusan

Kimia
radioactive DNA Dr.  rer.  nat.  Syafrizayanti  

27/11/17 31

Jurusan
DNA Ligase Is Activated by NAD+ or ATP Kimia
Dr.  rer.  nat.  Syafrizayanti  

E. coli DNA ligase, a 77-kD

27/11/17 32

16  
 27/11/17  

Jurusan
E. coli DNA ligase catalyzes a three-step reaction Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 33

Can small part of DNA Pol I Jurusan

Kimia
synthesize DNA? Dr.  rer.  nat.  Syafrizayanti  

The Klenow Fragment

euro-bio-net.net

27/11/17 34

17  
 27/11/17  

Jurusan
Replication Terminates at Specific Sites Kimia
Dr.  rer.  nat.  Syafrizayanti  

Note:
oriC is directly opposite the termination
region on the E. coli chromosome

•  The arrest of replication fork motion at Ter sites requires the action of Tus protein
a 309-residue monomer that is the product of the tus gene (for terminator
utilization substance)
•  Tus protein specifically binds to a Ter site, where it prevents strand displacement
by DnaB helicase, thereby arresting replication fork advancement.

27/11/17 35

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 36

18  
 27/11/17  

Jurusan
Overall Prokaryotic DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 37

Jurusan
Replication must be very accurate Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  First level of proof

reading:
The active site only fits
A=T and G=C base
pairs

•  One mistake is
made every 104 to
105 base pairs

27/11/17 38

19  
 27/11/17  

Jurusan
DNA Is Replicated with High Fidelity Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  Only one mispairing occurs per 108

to 1010
base pairs replicated.
•  This corresponds to 1 error per 1000 bacteria per generation

How to maintain such replication accuracy?

•  Cells maintain balanced levels of dNTPs
•  The polymerase reaction itself has extraordinary fidelity because it
occurs in two stages, control dNTP activation and polymerization
initiation
•  The 3’ à 5’ exonuclease functions of Pol I and Pol III detect and
eliminate the occasional errors made by their polymerase functions
•  A remarkable set of enzyme systems in all cells repairs residual
errorsin the newly synthesized DNA as well as any damage

27/11/17 39

Jurusan
CHECK YOUR UNDERSTANDING Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  Summarize the functions of the following

proteins in E. coli DNA replication: DNA
polymerase I, DNA polymerase III, DnaA,
helicase, SSB, primase, the sliding clamp, clamp
loader, DNA ligase, Tus, and topoisomerase.
•  How many sliding clamps and clamp-loading
events are required for synthesis of the leading
and lagging strands?
•  Explain the factors that contribute to the high
fidelity of DNA replication

27/11/17 40

20  
 27/11/17  

Jurusan
Eukaryotic DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

Learning •  Understand that eukaryotic DNA replication requires DNA

objectives   polymerases with different degrees of accuracy and
processivity.
•  Understand that pre-replication complexes assemble at
multiple origins distributed throughout the eukaryotic
genome.
•  Understand that telomerase uses an RNA template to
extend the 3_ ends of eukaryotic chromosomes.  

27/11/17 41

Jurusan
Eukaryotic DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  Eukaryotic DNA replication requires DNA

polymerases with different degrees of accuracy
and processivity.

•  Pre-replication complexes assemble at multiple

origins distributed throughout the eukaryotic
genome.
•  Telomerase uses an RNA template to extend
the 3_ ends of eukaryotic chromosomes.  

27/11/17 42

21  
 27/11/17  

Jurusan
When does replication take place? Kimia
Dr.  rer.  nat.  Syafrizayanti  

The vast majority of

DNA synthesis occurs
during S phase of the
cell cycle

27/11/17 43

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

Eukaryotic
DNA polymerases

27/11/17 44

22  
 27/11/17  

Jurusan
Eukaryotes Use Several DNA Polymerases Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 45

Jurusan
Eukaryotic DNA Polymerases Kimia
Dr.  rer.  nat.  Syafrizayanti  

Eukaryotic cells have counterparts for each of prokaryotic

enzymes plus some additional ones

E.coli Mammalian Function

I α Gap filling and synthesis of lagging strand
II ε DNA proofreading and repair
β DNA repair
γ mitochondrial synthesis
III δ Processive, Leading strand synthesis

27/11/17 46

23  
 27/11/17  

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 47

Different polymerases can hold on to Jurusan

Kimia
the DNA for longer or shorter time Dr.  rer.  nat.  Syafrizayanti  

q How long the polymerase

can hold on to the DNA
without falling of is called
processivity
q A polymerase with low
processivity falls of the
DNA easily and is not good
for replication on an entire
genome
Hang in there kiddo!

getrichslowly.org
27/11/17 48

24  
 27/11/17  

Jurusan
Eukaryotic DNA Is Replicated from Multiple Origins Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 49

Jurusan
Eukaryotic DNA Is Replicated from Multiple Origins Kimia
Dr.  rer.  nat.  Syafrizayanti  

Electron micrograph of a fragment of replicating Drosophila DNA

27/11/17 50

25  
 27/11/17  

Jurusan
Additional Enzymes Participate in Eukaryotic DNA Replication Kimia
Dr.  rer.  nat.  Syafrizayanti  

MCM: a helicase
RPA: equivalent of the bacterial SSB
PCNA : sliding clamp

27/11/17 51

Jurusan
Replication of a linear chromosome Kimia
Dr.  rer.  nat.  Syafrizayanti  

•  Leading strand synthesis

can proceed to the end of the
chromosome
•  DNA polymerase cannot
synthesize the extreme 5’ end
of the lagging strand
because it can only extend an
RNA primer
•  Removal of the primer and degradation of the
remaining single-stranded extension would cause the
chromosome to shorten with each round of
replication.

27/11/17 52

26  
 27/11/17  

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

Removal of RNA
primers in eukaryotes

27/11/17 53

Jurusan
Telomerase Extends Chromosome Ends Kimia
Dr.  rer.  nat.  Syafrizayanti  

Telomeres Are Built from an RNA Template

Mechanism for the synthesis of telomeric

DNA by Tetrahymena telomerase.

•  Unsual structure
•  Telomeric DNA consists of
1000 or more tandenm repeat
of a short G rich sequence on
the ending 3’-ending strand of
each chromosome end
ü TTGGGG in protozoan
Tetrahymena
ü TTAGGG in human
27/11/17 54

27  
 27/11/17  

Jurusan
Telomere Shortening As the Timekeeper of Cells Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 55

Jurusan
DNA Damage and Repair Kimia
Dr.  rer.  nat.  Syafrizayanti  

Learning •  Understand that DNA is susceptible to damage

Objectives from a variety of sources.
•  Understand that mutagenicity, which is related to
carcinogenicity, can be tested.
•  Understand that some DNA damage can be
repaired by the action of a single enzyme

27/11/17 56

28  
 27/11/17  

Jurusan
Repair Enzymes for DNA damage Kimia
Dr.  rer.  nat.  Syafrizayanti  

Environmental and
Chemical Agents
physically damage DNA.
and Generate Mutations

•  ultraviolet light
•  ionizing radiation
•  certain chemical agents

27/11/17 57

DNA Repair Earned the Nobel Prize in Jurusan

Kimia
Chemistry Dr.  rer.  nat.  Syafrizayanti  

Tomas Lindahl and DNA Decay Aziz Sancar and Repairing UV Damage
Base excision repair Nucleotide Excision Repair

Paul Modrich and Mismatch Repair

Mismatch Repair

27/11/17 58

29  
 27/11/17  

DNA repair in humans have linked many human diseases with Jurusan
decreased repair Kimia
Dr.  rer.  nat.  Syafrizayanti  

Molecular Biology of The Cell, 6th ed

27/11/17 59

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 60

30  
 27/11/17  

The most frequent spontaneous chemical reactions that create Jurusan

serious DNA damage in cells. Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 61

How chemical modifications of nucleotides Jurusan

Kimia
produce mutations Dr.  rer.  nat.  Syafrizayanti  

27/11/17 62

31  
 27/11/17  

Jurusan
Two major DNA repair pathways Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 63

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 64

32  
 27/11/17  

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 65

Jurusan
Kimia
Dr.  rer.  nat.  Syafrizayanti  

27/11/17 66

33