Lecture 2 DNA replication process and events.ppt

DNA replication

1. Molecular BiologyMolecular Biology
Lecture 2Lecture 2
DNA ReplicationDNA Replication
Dr. Emad Ibrahim OsmanDr. Emad Ibrahim Osman

2. DNA ReplicationDNA Replication
All cells undergo a division cycle during All cells undergo a division cycle during
their life span. their life span.
Some cells are continually dividing (e.g. Some cells are continually dividing (e.g.
haemopoietic cells), others divide a haemopoietic cells), others divide a
specific number of times until cell death specific number of times until cell death
occurs.occurs.
During the process of cell division During the process of cell division
everything within the cell must be everything within the cell must be
duplicated in order to ensure the survival duplicated in order to ensure the survival
of the two resulting daughter cells. of the two resulting daughter cells.

3. G1
M
G2
S
The Cell Life CycleThe Cell Life Cycle
Gap 1 - Doubling
of cell size.
Regular cellular
activities.
transcription and
translation etc.
Synthesis of DNA -
Regular cell
activities cease and
a copy of all nuclear
DNA is made
Gap 2 - Final
preparation for
division
Mitosis - Cell
division

4. DNA replication: DNA replication: It is the synthesis of a It is the synthesis of a
new DNA from the originally existing new DNA from the originally existing
""templatetemplate“ DNA. “ DNA.
It makes an identical copy (It makes an identical copy (duplicateduplicate) )
of the existing oneof the existing one

5. 
When the two strands of the DNA When the two strands of the DNA
double helix are separated, each can double helix are separated, each can
serve as a template for the replication serve as a template for the replication
of a new complementary strand.of a new complementary strand.

This produces two daughter This produces two daughter
molecules, each of which contains two molecules, each of which contains two
DNA strands with an antiparallel DNA strands with an antiparallel
orientationorientation

This process is called This process is called semiconservative semiconservative
replicationreplication

8. 
When the two strands of the When the two strands of the
DNA double helix are separated, DNA double helix are separated,
each can serve as a template for each can serve as a template for
the replication of a new the replication of a new
complementary strand.complementary strand.

This process is called This process is called
semiconservative replicationsemiconservative replication

This produces two daughter This produces two daughter
molecules, each of which molecules, each of which
contains two DNA strands with contains two DNA strands with
an antiparallel orientationan antiparallel orientation

9. DNA replication in DNA replication in
prokaryotesprokaryotes

The reactions of DNA replication were The reactions of DNA replication were
first known from studies of the first known from studies of the
bacterium Escherichia coli (E. coli), and bacterium Escherichia coli (E. coli), and
this represent the process in this represent the process in
prokaryotes.prokaryotes.

10. Processes in DNA synthesisProcesses in DNA synthesis
1) Separation of the two complementary 1) Separation of the two complementary
DNA strandsDNA strands
2)Formation of the replication fork2)Formation of the replication fork
3) Direction of DNA replication3) Direction of DNA replication
4) RNA primer4) RNA primer
5) Chain elongation5) Chain elongation
6) Excision of RNA primers and their 6) Excision of RNA primers and their
replacement by DNAreplacement by DNA
7) Ligation of nicks by DNA ligase7) Ligation of nicks by DNA ligase

11. 1) Separation of the DNA strands1) Separation of the DNA strands

In prokaryotic organisms, DNA In prokaryotic organisms, DNA
replication begins at a single, uniquereplication begins at a single, unique

nucleotide sequence, a site called the nucleotide sequence, a site called the
origin of replication (Ori)composed origin of replication (Ori)composed
almost exclusively of AT base pairs almost exclusively of AT base pairs
that facilitate melting.that facilitate melting.

In eukaryotes, replication begins at In eukaryotes, replication begins at
multiple sites along the DNA helixmultiple sites along the DNA helix

13. 22) Formation of the replication fork) Formation of the replication fork

As the two strands unwind and As the two strands unwind and
separate, they form a “V” separate, they form a “V”

This region is called the replication This region is called the replication
forkfork

Replication of dsDNA is bidirectionalReplication of dsDNA is bidirectional
—that is, the replication forks move —that is, the replication forks move
in opposite directions from the in opposite directions from the
origin, generating a replication origin, generating a replication
bubble bubble 1. 1.

15. 
Proteins required for DNA strand Proteins required for DNA strand
separation: Initiation of DNA separation: Initiation of DNA
replication requires the recognition replication requires the recognition
of the origin of replication by a group of the origin of replication by a group
of proteinsof proteins

These proteins include the following:These proteins include the following:
a. a. DnaA proteinDnaA protein: DnaA protein binds to : DnaA protein binds to
specific nucleotide causing short, specific nucleotide causing short,
tandemly arranged AT-rich regions in tandemly arranged AT-rich regions in
the origin to melt.the origin to melt.

16. 
b. DNA helicasesb. DNA helicases: These enzymes bind : These enzymes bind
to ssDNA near the replication fork, to ssDNA near the replication fork,
and then unwinding the double helix. and then unwinding the double helix.
Helicases require energy provided by Helicases require energy provided by
ATPATP

17. 
c. Single-stranded DNA-binding (SSB) c. Single-stranded DNA-binding (SSB)
proteinsproteins: These proteins bind to the : These proteins bind to the
ssDNA generated by helicases ssDNA generated by helicases

These proteins keep the two strands These proteins keep the two strands
of DNA separated in the area of the of DNA separated in the area of the
replication origin, also protect the replication origin, also protect the
DNA from nucleases that degrade DNA from nucleases that degrade
ssDNA.ssDNA.

19. 
During unwinding the accumulating During unwinding the accumulating
positive supercoils interfere with positive supercoils interfere with
further unwinding of the double helixfurther unwinding of the double helix

DNA topoisomerasesDNA topoisomerases: These enzymes : These enzymes
reversibly cut strands of the double reversibly cut strands of the double
helix. helix.

They have both nuclease (strand-They have both nuclease (strand-
cutting) and ligase (strand-resealing) cutting) and ligase (strand-resealing)
activities.activities.

ciprofloxacin target bacterial topoisomerase (DNA ciprofloxacin target bacterial topoisomerase (DNA
Gyrase)Gyrase)

20. 3) Direction of DNA replication3) Direction of DNA replication

The DNA polymerases responsible for The DNA polymerases responsible for
copying the DNA templates are only copying the DNA templates are only
able to “read” the parental nucleotide able to “read” the parental nucleotide
sequences in the 3' 5' direction, and
→
sequences in the 3' 5' direction, and
→
they synthesize the new DNA strands they synthesize the new DNA strands
only in the 5' 3' (antiparallel)
→
only in the 5' 3' (antiparallel)
→
direction. direction.

The replication process accomplished The replication process accomplished
by different mechanism in each strandby different mechanism in each strand

21. 1. 1. Leading strandLeading strand: The strand that is : The strand that is
being copied in the direction of the being copied in the direction of the
advancing replication fork is called the advancing replication fork is called the
leading strand and is synthesized leading strand and is synthesized
continuously.continuously.
2. 2. Lagging strandLagging strand: The strand that is : The strand that is
being copied in the direction away being copied in the direction away
from the replication fork is synthesized from the replication fork is synthesized
discontinuously, with small fragments discontinuously, with small fragments
of DNA termed of DNA termed Okazaki fragmentsOkazaki fragments

22. 4) RNA primer4) RNA primer

DNA polymerases cannot initiate DNA polymerases cannot initiate
synthesis of a complementary strand synthesis of a complementary strand
of DNA on a totally single-stranded of DNA on a totally single-stranded
template.template.

PrimasePrimase: A specific RNA polymerase, : A specific RNA polymerase,
called primase (called primase (DnaGDnaG), synthesizes ), synthesizes
the short stretches of RNAthe short stretches of RNA

24. 5) Chain elongation5) Chain elongation

Prokaryotic (and eukaryotic) DNA Prokaryotic (and eukaryotic) DNA
polymerases elongate a new DNA polymerases elongate a new DNA
strand by adding strand by adding
deoxyribonucleotides, one at a time, deoxyribonucleotides, one at a time,
to the 3'-end of the growing chain.to the 3'-end of the growing chain.

DNA chain elongation is catalyzed by DNA chain elongation is catalyzed by
DNA polymerase IIIDNA polymerase III. .

25. 
To ensure replication fidelity, DNA To ensure replication fidelity, DNA
polymerase III has, in addition to its polymerase III has, in addition to its
5' 3' polymerase activity, a
→
5' 3' polymerase activity, a
→
“proofreading” activity “proofreading” activity
(3' 5'exonuclease)
→
(3' 5'exonuclease)
→

26. 6) Excision of RNA primers and their 6) Excision of RNA primers and their
replacement by DNAreplacement by DNA

DNA polymerase III continues to DNA polymerase III continues to
synthesize DNA on the lagging strand synthesize DNA on the lagging strand
until it is blocked by proximity to an until it is blocked by proximity to an
RNA primer. RNA primer.

When this occurs, the RNA is excised When this occurs, the RNA is excised
and the gap filled by and the gap filled by DNA polymerase DNA polymerase
II..

27. 
In addition to having the 5' 3'
→
In addition to having the 5' 3'
→
polymerase activity that synthesizes polymerase activity that synthesizes
DNA, and the 3' 5' exonuclease
→
DNA, and the 3' 5' exonuclease
→
activity that proofreads the newly activity that proofreads the newly
synthesized DNA chain like DNA synthesized DNA chain like DNA
polymerase III, polymerase III,

DNA polymerase I also has a 5' 3'
→
DNA polymerase I also has a 5' 3'
→
exonuclease activity that is able to exonuclease activity that is able to
hydrolytically remove the RNA primer.hydrolytically remove the RNA primer.

29. 7) Ligation by DNA ligase7) Ligation by DNA ligase

The final The final phosphodiesterphosphodiester linkage linkage
between the 5'-phosphate group on between the 5'-phosphate group on
the DNA chain synthesized by DNA the DNA chain synthesized by DNA
polymerase III and the 3'-hydroxyl polymerase III and the 3'-hydroxyl
group on the chain made by DNA group on the chain made by DNA
polymerase I is catalyzed by polymerase I is catalyzed by DNA DNA
ligaseligase

30. Nick translation of DNA. DNA polymerase extends the 3 end of a
nick in double-stranded DNA with newly synthesized strand (gray)
while digesting the original strand from the 5 end. After
polymerization, the nick is closed by DNA ligase.

31. DNADNA
polymerase-Ipolymerase-I..
DNADNA
polymerase-IIpolymerase-II
DNADNA
polymerase-IIIpolymerase-III..
E.ColiE.Coli
DNA polymerasesDNA polymerases

32. DNA polymerase IDNA polymerase I
It is the most abundant replicating enzyme It is the most abundant replicating enzyme
in in E. coliE. coli
Removes the primers from the lagging Removes the primers from the lagging
strandstrand
Ensure the fidelity of replication through the Ensure the fidelity of replication through the
repair of damaged and mismatched DNA repair of damaged and mismatched DNA

33. DNA polymerase IIDNA polymerase II
It’s primary role is to ensure the It’s primary role is to ensure the
fidelity of replication through the fidelity of replication through the
repairrepair of damaged and mismatched of damaged and mismatched
DNA (proof-reading)DNA (proof-reading)

34. DNA polymerase IIIDNA polymerase III
This enzyme is much less abundant This enzyme is much less abundant
than pol I, however, its activity is than pol I, however, its activity is
nearly 100 times that of pol I.nearly 100 times that of pol I.
It is responsible for the bulk replication It is responsible for the bulk replication
of the of the E. coliE. coli genome, genome,

35. Eukaryotic DNA synthesisEukaryotic DNA synthesis

The process of eukaryotic DNA The process of eukaryotic DNA
replication Similar to that of replication Similar to that of
prokaryotic DNA synthesis. prokaryotic DNA synthesis.

Some differences, such as the Some differences, such as the
multiplemultiple originsorigins of replication in of replication in
eukaryotic cells versus single origins eukaryotic cells versus single origins
of replication in prokaryotes.of replication in prokaryotes.

In contrast, RNA primers are removed In contrast, RNA primers are removed
by by RNase H RNase H rather than by a DNA rather than by a DNA
polymerase.polymerase.

36. Eukaryotic DNA polymerasesEukaryotic DNA polymerases

At least five key eukaryotic DNA At least five key eukaryotic DNA
polymerases have been identified and polymerases have been identified and
categorized on the basis of molecular categorized on the basis of molecular
weight, cellular location, sensitivity to weight, cellular location, sensitivity to
inhibitors, and the templates or inhibitors, and the templates or
substrates on which they act. substrates on which they act.

There have been 5 distinct eukaryotic There have been 5 distinct eukaryotic
DNA polymerases identified, DNA polymerases identified, αα, ß, , ß, γγ, , δδ
and and εε. .

38. ►The ability of DNA polymerases to The ability of DNA polymerases to
replicate DNA requires a number of replicate DNA requires a number of
additional accessory proteins. additional accessory proteins.
►The combination of polymerases with The combination of polymerases with
several accessory proteins yields several accessory proteins yields DNA DNA
polymerase holoenzyme. polymerase holoenzyme.

39. Post-Replicative Modification of Post-Replicative Modification of
DNA, MethylationDNA, Methylation
►One of the major post-replicative One of the major post-replicative
reactions that modifies the DNA is reactions that modifies the DNA is
methylationmethylation..
►The sites of natural methylation of The sites of natural methylation of
eukaryotic DNA is always on eukaryotic DNA is always on cytosinecytosine
residues.residues.

40. DNA MethylationDNA Methylation
►The cytidine is methylated at the 5 The cytidine is methylated at the 5
position of the pyrimidine ring position of the pyrimidine ring
generating generating 5-methylcytidine5-methylcytidine
►Methylation also occurs in DNA of Methylation also occurs in DNA of
prokaryotic cells. prokaryotic cells.

41. oThe function of this methylation is to The function of this methylation is to
prevent degradation of host DNA in the prevent degradation of host DNA in the
presence of presence of restriction endonucleasesrestriction endonucleases
oThe role of this system in prokaryotic The role of this system in prokaryotic
cells is to destroy invading viral DNAs. cells is to destroy invading viral DNAs.
DNA MethylationDNA Methylation

42. 1.1.DNA replication must possess a very DNA replication must possess a very
high degree of high degree of fidelityfidelity. .
2.2.The entire process of DNA replication The entire process of DNA replication
is complex and involves is complex and involves multiple multiple
enzymatic enzymatic activities. activities.

43. 3.3.Occurs during the Occurs during the S phase S phase of the cell of the cell
cycle cycle
4.4.Starts at Starts at specific sites specific sites in DNAin DNA
5.5.Involves Involves several proteins several proteins and enzymes.and enzymes.

44. 6.6.Occurs Occurs onceonce during a cell cycle. during a cell cycle.
7.7.Involves the Involves the whole genomewhole genome..
8.8.Is Is template dependanttemplate dependant, directed by base , directed by base
pairing.pairing.
9.9.Requires Requires primerprimer for initiation for initiation

45. 10.10. Is Is semiconservativesemiconservative..
11.11. Proceeds Proceeds in 5in 533 direction. direction.
12.12. Occurs Occurs bidirectionallybidirectionally and simultaneously in both and simultaneously in both
strandsstrands
13.13. DiscontinuousDiscontinuous in the lagging strand in the lagging strand