Beruflich Dokumente
Kultur Dokumente
1
Learning Objectives
After reading this chapter you should be able to:
Describe the different types of nucleotides & their
function
Compare and contrast the structure and biosynthesis of
purines and pyrimidines
Highlight the differences between de novo and salvage
pathways.
Describe the degradative pathways for purine and
pyrimidine nucleotides
Explain the metabolic basis and therapy for classic
disorders in nucleotide metabolism: Lesch-Nyhan
syndrome, gout and SCIDS.
Describe the different chemotherapeutic targets in 2
nucleotide metabolisms.
3
Baynes and Dminiczak: Medical biochemistry 2e or 3e
1. Introduction: nucleotide structure and function
Nucleotides are building blocks of nucleic acids
They are formed from three components:
a nitrogenous base
a five-carbon sugar
a phosphate moiety
without a phosphate group they are called
nucleoside
4
Nucleoside and Nucleotide
5
Nitrogenous Bases
There are two types bases:
Purine: guanine and adenine
Pyrimidine: thymine, cytosine, & uracil
6
Other/minor /modified bases
7
Sugars Phosphate Groups
Mono-, di- or triphosphates
D-Ribose and 2- Phosphates can be bonded to either
Deoxyribose C3 or C5 atoms or both of the sugar
pyrimidine OR purine
N-b-glycosyl
bond
Ribose
or
2-deoxyribose
9
10
Biological functions of nucleotides
- alanine
12
13
De novo synthesis of purines
Site: in cytosol of liver (mainly)
Basic pathway for biosynthesis of purine ribonucleotides.
1. comprising of 11 reactions and consumes energy(~ 6ATP)
2. Starts from ribose-5-phosphate which is converted into PRPP
3. Ribose-5-phosphate derived from the pentose phosphate pathway.
4. PRPP is also a precursor in the biosynthesis of pyrimidines and
the amino acids tryptophan and histidine
5. The first intermediate formed with a complete purine ring is
inosinate (IMP)
6. Once IMP formed, it is rapidly converted to AMP and GMP
14
sources of purine bases
(N9)
(C4,C5,N7)
N10-
Formyltetrah
ydrofolate
N10-
Formyltetrahy
drofolate
15
John M. Buchanan, 19172007
STEPS: Purine Nucleotide Synthesis (see) O
COO
OOC C
N HC N N
C4 Aspartate C4
ADP
C
5
N
CH + ATP 8 + Pi CH2
H
C
5
N
CH
H2N COO
Ribose-5-Phosphate
N1
ATP Carboxyamidoimidazole Ribotide (CAIR) 5-Aminoimidazole-4-(N-succinylocarboxamide)
Ribose
1 Phosphate AIR
ADP + Pi
ribotide (SAICAR)
AMP
Pyrophosphokinase Car boxylase
7
ATP
+HCO3
Fumarate
O
Adenylosuccinate
Lyase
N 9
C6 C
2-
O3P O CH2
O
H HC 4
O O H2N N
H H CH C4
5
H O P O P O C CH
OH N C
5
OH
H2N N
O O H2N
Ribose-5-Phosphate
Ribose-5-Phosphate
5-Aminoimidazole Ribotide (AIR)
5-Phosphoribosyl--pyrophosphate (PRPP) 5-Aminoimidazole-4-carboxamide
ADP + Pi ribotide (AICAR)
AIR
Glutamine
+ H2O Amidophosphoribosyl
Synthetase
6
ATP
N10-Formyl-
THF 10
2
Glutamate
Transferase
H O THF
AICAR
Transformylase
+ PPi N
H2C CH C
H2N N
2-
O3P O CH2
O
NH2
b
N9 C O N3 C4
CH
H
H H
H
HN NH
O C NH
C
5
N C2
OH OH H
Ribose-5-Phosphate Ribose-5-Phosphate
b-5-Phosphoribosylamine (PRA)
Formylglycinamidine ribotide (FGAM) 5-Formaminoimidazole-4-carboxamide
Glycine ADP + ribotide (FAICAR)
+ ATP Glutamate + Pi
FGAM
3
ADP
GAR Synthetase Synthetase
5 ATP +
H2O
IMP
Cyclohydrolase
+ Pi
H
Glutamine +
H2O O
11
hypoxanthine
C4,C5,N7 H 2C NH2 10
N -Formyl-THF THF H 2C
N
CH HN
C
C
N
4
O C CH
HC C5
2-
C O N
NH N
O3P O CH2
H
O
H
O NH
C8 2-
O3P O CH2
H
O
H
H
OH
H GAR Transformylase Ribose-5-Phosphate H H 16
OH OH OH
Humans unaffected by
sulfonamides.
Rxn8&9
18
IMP dehydrogenase& MPA
Mycophenolic acid (MPA) immunosuppressant drug that
inhibits IMP dehydrogenase activity
IMP dehydrogenase is essential to the immune response(as
B and T lymphocytes proliferation) to generate the
guanosine nucleotides they need to proliferate.
Moreover, certain cancer cells have increased IMP
dehydrogenase activity.
Hence, IMP dehydrogenase is a target for both
immunosuppressive therapy and cancer chemotherapy.
Indeed, MPA is in clinical use to prevent the rejection of
transplanted kidneys.
19
ADP, ATP, GDP and GTP biosynthesis
20
Regulation
ribose phosphate pyrophosphokinase
Gln:PRPP amidotransferase
ADA deficiency
Lesch-Nyhan syndrome
There is a defect or lack of the HGPRT enzyme
Sex-linked metabolic disorder: only males
the rate of purine synthesis is increased about 200-
fold
Loss of HGPRT leads to elevated PRPP levels and
stimulation of de novo purine synthesis.
purine bases cannot be salvaged & instead, degraded
forming excessive amounts of uric acid.
uric acid level rises and there is gout
In addition there are mental aberrations
patients will self-mutilate by biting lips and fingers off
24
Lesch-Nyhan syndrome
25
The Purine Nucleotide Cycle
Plays an important metabolic role
in skeletal muscle.
An increase in muscle activity
requires an increase in the activity
of the citric acid cycle.
Muscles, however, lack most
of the enzymes that catalyze
these anaplerotic (filling up)
reactions in other tissues.
So muscle relies on AMP
deaminase for this purpose
Has net effect of
deaminating aspartate to
26
yield fumarate& ammonia
Formation of deoxyribonucleotide
Formation of deoxyribonucleotide involves the reduction
of the sugar moiety of ribonucleoside diphosphates (ADP,
GDP, CDP or UDP) by ribonucleotide reductase(RNR).
Deoxyribonucleotide synthesis at the nucleoside
diphosphate(NDP) level.
dNTPs Are Produced by Phosphorylation of dNDPs
P P O CH2 O Base P P O CH2 O Base
ribonucleotide
reductase
Mg2+
OH OH H2O OH H
thioredoxin SH thioredoxin S
NDP dNDP
SH S ATP
N=A, G, C, U
FAD kinase
NADP + NADPH + H+
thioredoxin 27ADP
reductase
RNR and hydroxyurea
Ribonucleotide reductase
28
Summary of purine biosynthesis
29
De novo synthesis pyrimidine bases
Shorter pathway than for purines
Pyrimidine ring is made first, then attached to
ribose-phosphate (unlike purine biosynthesis)
Only few precursors (aspartate and glutamine, plus
HCO3-) contribute to the 6-membered ring
Requires 6 steps (instead of 11 for purine)
The first product is UMP (uridine monophosphate)
30
Source of pyrimidine atoms
C
4
Gln N3 5C
Asp
CO2 C2 6C
1
N
31
O
2 ATP + HCO3- + Glutamine + H2O Pyrimidine Synthesis
C
HN CH
O
2 ADP +
Glutamate + Carbamoyl C C
Pi
Phosphate C O N
Synthetase II PRPP PPi
HN CH COO
2-
NH2 O3P O CH2
O
Orotate Phosphoribosyl b
C C Transferase
H H
O C O N H H
H COO OH OH
C
C CH
HN CH2 N
HO C H2O O
CH2 2-
O3P O CH2
NH2 C CH O
N H H b
O
C CH H COO H
Dihydroorotase H
O N OH OH
H COO Dihydroorotate 32
Uridine Monophosphate
(UMP)
Carbamoyl Aspartate
Contd
Leflunomide:
Inhibitor of dihydroorotate dehydrogenase (DHODH)( step 4)
used for the treatment of rheumatoid arthritis.
It attenuates this autoimmune disease by blocking
pyrimidine biosynthesis in T lymphocytes, thereby
reducing their inappropriate proliferation.
Inside DHODH)
33
Orotic aciduria
Results from a deficiency in the bifunctional enzyme UMP
synthase enzyme(Orotate phosphoribosyl Transferase(step
5) & orotidylate decarboxylase (step 6))
Treatment: the administration of uridine and/or cytidine.
The UMP is then formed from them.
Then inhibits carbamoyl phosphate synthetase II so as to
attenuate the rate of orotic acid synthesis.
Characterized by the excretion of large amounts of orotic
acid in the urine, retarded growth, and severe anemia.
34
3. UTP and CTP biosynthesis
NMkinase NDkinase
UMP UDP UTP
(amination)
O
NH3 O N 5 10
N
N , N -CH2-FH4 FH2
d R 5' P d R 5' P
H2O
dUMP FH2 dTMP
dCMP NADPH ATP
reductase
+ H+ ADP
FH4 dTDP
NADP + 36
ATP
ADP dTTP
Antifolates and Anticancer Agents
NH2 R O COOH
N
N CH2 N C NH C CH2 CH2 COOH
H
H2N N N
RH AP RCH3 TXT MtX
OH H O COOH
N
N CH2 N C NH C CH2 CH2 COOH
H
H2N N N
folic acid
37
FdUMP: Potent Antitumor Agent
Is an irreversible inhibitor of thymidylate synthase
The strategic position of thymidylate synthase in
DNA biosynthesis has led to the clinical use of
FdUMP as an antitumor agent.
Rapidly proliferating cells, such as cancer cells,
require a steady supply of dTMP in order to
survive and are therefore killed by treatment with
FdUMP.
5-Fluorouracil and 5-fluorodeoxyuridine are also
effective antitumor agents since they are converted
to FdUMP through salvage reactions.
38
Antifolates Are Anticancer Agents
Inhibition of dihydrofolate reductase(DHFR ) not only prevents dTMP
synthesis, but also blocks all other THF-dependent biological
reactions such as the synthesis of purines, methionine, and indirectly,
histidine.
DHFR therefore offers an attractive target for chemotherapy.
Methotrexate (amethopterin), aminopterin, and trimethoprim are
DHF analogs that irreversibly bind to DHFR with an 1000-fold
greater affinity than does DHF.
These antifolates are effective anticancer agents, particularly against
childhood leukemias.
A low dose of methotrexate is also effective in the treatment of
rheumatoid arthritis, inhibiting immune system activity and thus
decreasing inflammation.
Trimethoprim binds much more tightly to bacterial DHFRs than to
those of mammals and is therefore a clinically useful antibiotic.39
pyrimidine Salvage pathways
Pyrimidine phosphorylase
Thymidine phosphrylase
deoxycytidine kinase
deoxycytidine + ATP dCMP + ADP
pyrimidine phosphate
uracil ribosyltransferase UMP
thymine + PRPP dTMP +41 PPi
orotic acid OMP
Summary of pyrimidine biosynthesis
42
Nucleotide Degradation
1. purinenucleotide degradation
2. pyrimidine nucleotide degradation
43
1. Purine Nucleotide Degradation
ADA deficiency
Allopurinol
44
ADA deficiency
Autosomal recessive disorder
Causes severe combined immunodeficiency disease(SCID)
High activity in lymphocytes
Deficiency leads to overwhelming infection.
In the absence of active ADA, deoxyadenosine is
phosphorylated to yield high levels of dATP that are
50-fold greater than normal.
This high concentration of dATP inhibits
ribonucleotide reductase, thereby preventing the
synthesis of the other dNTPs, choking off DNA
synthesis and thus cell proliferation.
45
Treatment: polyethylene glycol-ADA (PEG-ADA)
Uric acid
Uric acid is the excreted end product of purine catabolism
in primates, birds, and some other animals.
The rate of uric acid excretion by the normal adult human
is about 0.6 g/24 h, arising in part from ingested purines
and in part from the turnover of the purine nucleotides of
nucleic acids.
The normal concentration of uric acid in the serum of
adults is in the range of 3-7 mg/dl.
46
Gout
Gout is a disease characterized by elevated levels of uric
acid in body fluids.
Its most common manifestation is extremely painful
arthritic joint inflammation of sudden onset, most often
in the big toe (caused by deposition of nearly insoluble
crystals of sodium urate.
Sodium urate and/or uric acid may also precipitate in the
kidneys and ureters as stones, resulting in renal damage
and urinary tract obstruction.
Causes:
impaired uric acid excretion ( most common)
LeschNyhan syndrome
glucose-6-phosphatase deficiency (von Gierkes glycogen
47
storage disease)
The uric acid and the gout Hypoxanthine
In urine
Uric acid
Over 8mg/dl, in the plasma
Diabetese nephrosis
Gout, Urate crystallization
in joints, soft tissue, cartilage and kidney
48
49
Allopurinol a suicide inhibitor used to treat Gout
O O
C C H
N C
HN C HN C
CH N
HC C HC C
N N N
N H H
Hypoxanthine Allopurinol
Xanthine oxidase
Xanthine oxidase
50
2. Pyrimidine
degradation
51
Summery Points
Synthesis of Purine Nucleotides
De novo synthesis: Site, Characteristics, Element sources of purine
bases
Salvage pathway: definition, significance, enzyme, Lesch-Nyhan
syndrome
Formation of deoxyribonucleotide: NDP level
Antimetabolites of purine nucleotides:
Purine, Amino acid, and Folic acid analogs
Degradation of Purine Nucleotides
Uric acid, gout
Synthesis of Pyrimidine Nucleotides
De novo synthesis: Characteristics, Element sources of pyrimidine
bases
Salvage pathway
Antimetabolites of pyrimidine nucleotides 52
Catabolism of Pyrimidine Nucleotides
Molecular Genetics
&
Molecular Biology
Section outline
DNA, Genes, chromosome
o Historic and general overviews
o DNA discovery, structure and Packaging
DNA Replication (DNA synthesis)
RNA Structure, Transcription & RNA processing
The Genetic Code, Protein Synthesis and Protein
modification
Mutation and Repair
Regulation of gene expression
Molecular biology tools and techniques in diagnosis
and treatment of diseases
Genetics in clinical medicine
Genetic disorders accounts for > 10% of pediatric admissions
and childhood mortality.
Virtually every medical condition has a genetic component.
many common disorders (HTN, CVD, asthma, DM, MI & so
many others significantly influenced by the genetic background.
better understanding of the genetic basis of these disease will
impact on their prevention.
almost all cancer has a genetic basis
Study of genetics enhances our understanding of disease
etiology and pathogenesis as well as its treatment & diagnosis
detection of infectious pathogens ( e.g. PCR)
Gene and Protein therapy
Precision medicine???
Forensic analysis
Nucleic acids
In 1869 Friedrich Miescher isolated what he called nuclein
from the nuclei of pus cells
Nuclein was shown to have acidic properties, hence it
became called nucleic acid
Two major classes of nucleic acids:
Deoxyribonucleic acid (DNA): carrier of genetic
information
Ribonucleic acid (RNA): an intermediate in the
expression of genetic information and other diverse roles
Nucleic Acids.
Function:
Genetic material
stores information
genes
blueprint for building proteins
transfers information
blueprint for new cells
DNA
blueprint for next generation
proteins
Genetic material
To fulfill its role, the genetic material must meet the
following criteria
1. Information: It must contain the information necessary to
make an entire organism
2. Transmission: It must be passed from parent to offspring
3. Replication: It must be copied in order to be passed from
parent to offspring
4. Variation: It must be capable of changes
To account for the known phenotypic variation in each
species
58
DNA as a genetic material: Experimental proofs
the identification of DNA as the genetic material involved
a series of outstanding experimental approaches
Frederick Griffiths Experiment with Streptococcus
pneumoniae (1928)
The Experiments of Avery, MacLeod &McCarty
(1940s)
Hershey & Chase blender experiment(1952)
Kinds of Genetic Elements
60
Structure
Polymers of Nucleic
of nucleotides Acids
(polynucleotides)
Nucleotides are linked together to form a linear strand of
RNA or DNA
In DNA two strands interact to form a double helix
Polynucleotides are covalently linked together by phosphodiester
bonds
A phosphate connects the 5 carbon of one nucleotide to the
3 carbon of another
The two strands are joined by the base pairs
Each base is paired with a specific partner: A is always paired
with T G is always paired with C
The bases are joined by hydrogen bonds, individually weak but
collectively strong
Composition of DNA and RNA
O O NH 2
5
N 6 H H
HOCH2 OH 4
O 1N
5
3N
7 5
4 1 H 8 2
H H 9 4 6 1
2
H H N 3
NH2 H O
N N
3 2
HO OH H H
D-Ribose (in RNA) Guanine (G) Cytosine (C)
63
The structure of nucleotides found in (a) DNA and (b) RNA
A, G, C or T A, G, C or U
O Base O Base
O P O CH2 O P O CH2
5 O 5 O
O 4 1 O 4 1
H H H H
H H H H
Phosphate 3 2
Phosphate 3 2
OH H OH OH
Deoxyribose Ribose
Deoxyribonucleotide
65
Ribonucleotide
Experimental works that Led to the Discovery of DNA
Structure
66
H
Linus Pauling C
C N
H O C
H C
N C C N
In the early 1950s, he C
O Carbonyl
oxygen
O
proposed that regions of C
H
H Amide
O N C hydrogen
protein can fold into a C N
secondary structure H H
O
C
C
C N
a-helix N C O
C
HO Hydrogen
C H bond
To elucidate this structure, O N C
C N
he built ball-and-stick H H
O C
C
models N C N
O
C
O
Helical
More than one strand
10 base pairs per
complete turn
68
Erwin Chargaffs Experiment
Chargaff analyzed the base composition of DNA isolated
from many different species
It was known that DNA contained the four bases: A, G, C
and T
The compelling observation was that
Percent of adenine = percent of thymine
Percent of cytosine = percent of guanine
This observation became known as Chargaffs rule
It was a crucial piece of evidence that Watson and Crick used to
elucidate the structure of DNA
69
An analysis of the base composition of DNA in different
species may reveal important features about the
structure of DNA
70
Some of Chargaffs Rules
1. DNA base composition varies between species.
2. DNA from different tissues of same species has the same
base composition.
3. Base composition of a species does not vary with age,
nutritional state, or change in environment.
4. No matter what species A = T and G = C and [purines] =
[pyrimidines] which is A+G = T+C
These rules made sense when applied to the Watson-Crick
DNA structure.
Watson and crick:1953
ds DNA on the
English 2 Pound
Coin (b) Original model of the DNA double helix
Watson and Crick
Familiar with all of these key observations, Watson and
Crick set out to solve the structure of DNA
They tried to build ball-and-stick models that
incorporated all known experimental observations
A critical question was how the two (or more strands)
would interact
An early hypothesis proposed that DNA strands interact through
phosphate-Mg++ cross-links(incorrect hypothesis)
73
Watson and Crick..
They went back to the ball-and-stick units
They then built models with the
Sugar-phosphate backbone on the outside
Bases projecting toward each other
They first considered a structure in which bases form
H bonds with identical bases in the opposite strand
i.e. A to A, T to T, C to C, and G to G
Model building revealed that this also was incorrect
They then realized that the hydrogen bonding of A to T
was structurally similar to that of C to G
So they built ball-and-stick models with AT and CG
interactions between the two DNA strands
These were consistent with all known data about DNA structure
74
Watson&Crick .
After that Watson and Crick postulated that:
1. DNA is a double helix where two helical DNA strands coiled around same
axis to form right-handed double helix
The strands of DNA comprising the double helix run in opposite
direction. i.e Strands are antiparallel.
The two DNA strands are not identical, but they are complementary.
2. The DNA double helix structure confirms the Chargaffs rules.
i.e. A bonded to T & C bonded to G
3. Hydrophilic backbone of alternating sugar/phosphate units of the
nucleotides are on outside of DNA molecule exposed to water
4. Hydrophobic bases (complementary base pairs) are stacked neatly inside
the molecule.
The hydrogen bond between the base pairs hold the double helix together
5. There are 10 base pairs per complete turn of the helix (3.4 nm)
6. The spatial relationship between the two strands creates a major groove and
a minor groove between the two strands
The Watson-Crick Structures
1.085nm
Phosphodiester bonds
Base pairing Hydrogen bonds
P
G C
P
P
C G
P
P
C G
P
P
A T
P
P
T A
P
P
T A
P
DNA VS RNA
Deoxyribonucleotide
79
Ribonucleotide
Structural forms of the double DNA Helix
10-6
Bacterial genome
The bacterial chromosome is found in a region called the
nucleoid (DNA+protein region)
Bacterial chromosomal DNA is usually a circular
molecule that is a few million nucleotides in length
Escherichia coli ~ 4.6 million base pairs
Haemophilus influenzae ~ 1.8 million base pairs
A typical bacterial chromosome contains a few thousand
different genes
Structural gene sequences (encoding proteins) account
for the majority of bacterial DNA
The non-transcribed DNA between adjacent genes are
termed intergenic regions
A few hundred
nucleotides in length
Cistron 1 Cistron 2
Keys:
4
Therefore the DNA must be compacted ~10 -fold.
0.2-2
m
Compaction level
in euchromatin
During interphase
most chromosomal Compaction level
regions are in heterochromatin
euchromatic
Heterochromatin vs Euchromatin
Compaction level of interphase chromosomes is
not uniform
Euchromatin
Less condensed regions of chromosomes
Transcriptionally active
Regions where 30 nm fiber forms radial loop domains
Heterochromatin
Tightly compacted regions of chromosomes
Transcriptionally inactive (in general)
Radial loop domains compacted even further
Histone Modifications
The N-terminal ends of these histones
can be acetylated, methylated, or phosphorylated.
DNA supercoiling
Supercoils are introduced into DNA when a duplex is twisted
in space around its own axis.
It is an important way to compact the chromosome
Supercoiling is involved in initiation of transcription,
replication, repair & recombination
Two types:
Negative supercoils:
twist the DNA about its axis in the opposite direction from
the clockwise turns of the right-handed (R-H) double helix.
Underwound (favors unwinding of duplex).
Has right-handed supercoil turns.
Positive supercoils:
twist the DNA in the same direction as the turns of the R-
H double helix
Overwound (helix is wound more tightly)
Has left-handed supercoil turns.
Topoisomerase:
The accumulating positive supercoils interfere with further
unwinding of the double helix.
To solve this problem, there is a group of enzymes called
DNA topoisomerases, which are responsible for removing
supercoils in the helix.
Two types:
Type I DNA topoisomerases:
Type II DNA topoisomerases
Type I DNA topoisomerases:
These enzymes reversibly cut one strand of the
double helix.
Each time a transient nick is created in one DNA
strand, the intact DNA strand is passed through the
break before it is resealed, thus relieving (relaxing)
accumulated supercoils.
Type I topoisomerases relax negative supercoils in E.
coli, and both negative and positive supercoils in
eukaryotic cells.
Topoisomerase II
Make transient breaks in both strands.
Its mechanism of action is to make a transient double
strand break, pass a duplex DNA through the break, and
then re-seal the break.
As a result, both negative and positive supercoils can be
relieved by this ATP-requiring process.
Topoisomerase found in bacteria( DNA gyrase) introduce
negative supercoils into relaxed circular DNA
This facilitates the future replication of DNA because the
negative supercoils neutralize the positive supercoils
introduced during opening of the double helix.
Topoisomerases as a therapeutic targets
Without topoisomerases, cells cannot replicate or package
their DNA, or express their genesand they die.
Inhibitors of topoisomerases are important pharmaceutical
agents, targeted at infectious agents and malignant cells.
1. bacterial topoisomerase inhibitors( DNA gyrase)
Quinolones such as ciprofloxacin
Novobiocin: blocks ATP binding
2. Chemotherapeutic agents used in cancer txt
Inhibitors of both type II human topoisomerases have been
developed as anticancer drugs.
Includes: aminoacridines, anthracenediones, ellipticines, and
epipodophyllotoxins, doxorubicin (Adriamycin), etoposide
(Etopophos)
DNA Replication
Ezra B. (M.Sc.)
Medical biochemistry Unit
DNA Replication
DNA replication is the process by
which the genetic material is copied
Bases for inheritance
Each cell must copy its entire
DNA before its division (transfer
of genetic material to daughter
cells)
occurs very quickly, very accurately
and at the appropriate time in the life
of the cell
extreme accuracy of DNA replication
is necessary in order to preserve the
integrity of the genome in successive
generations
Rate of synthesis
Bacteria= 1000 bases per second
Mammals= 100 bases per second
Models of DNA replication
Modes of DNA Replication
1. Theta replication(): A common type
of replication that takes place in circular
DNA, such as that found in E. coli and
other bacteria
2. Rolling-circle replication:
Takes place in some viruses
3
5
5
3
3
DNA gyrase is a topoisomerase II, which 5
breaks and reseals the DNA to introduce negative
supercoils ahead of the fork
Fluoroquinolone antibiotics target DNA gyrases in many
gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)
Formation of Replication forks
DNA molecules in the process of being replicated contain
Y/V-shaped junctions called replication forks.
Two replication forks are formed at each replication origin
Synthesis of DNA proceeds bi-directionally from the oric
The replication forks eventually meet at the opposite side
of the bacterial chromosome(This ends replication)
RNA Primer
DNA polymerases cannot initiate DNA synthesis
DNA polymerases can attach nucleotides only in the 5 to
3 direction
The enzyme RNA polymerase(primase) synthesizes an
RNA primer (10 to 12 nucleotides) that provides the free
3'-hydroxyl required by DNA polymerase III
These short RNA strands start, or prime, DNA synthesis
They are later removed and replaced with DNA polymerase
I
Initiation of DNA Synthesis
At the heart of the replication machine is an enzyme called
DNA polymerase, which synthesizes new DNA using one
of the old strands as a template.
All DNA polymerases require a primer, which is extended,
and a template strand, which is copied.
DNA polymerases can elongate an existing primer but
cannot initiate DNA synthesis.
This enzyme catalyzes the addition of nucleotides to the 3
end the RNA primer.
The energy for polymerization is provided by the incoming
nucleotide (NTP)
The reaction involves the formation of a phosphodiester
bond between the 3 end of the RNA/DNA chain and the 5-
phosphate group of the incoming nucleotide =>Chain
elongation occurs in the 5' to 3' direction
DNA Polymerases
In E. coli there are five proteins with polymerase activity
DNA pol I, II, III, IV and V
Normal replication
DNA pol III falls off the DNA template after a few dozen
DNA pol III uses the RNA primers to synthesize small DNA
-Upon completion of an
Okazaki fragment, the enzyme
releases the lagging template
strand
-Another loop is then formed
-This processed is repeated over
and over again
3 5
5 3
3 5 3 5
Prima
se Single
Laging Strand strand
5
Okazaki 5 binding
fragment 3 proteins
5
RNA
Prime
DNA rs 5
Polymer 3
ase
Helicase
Leading Strand
5
3
Keep the parental
Summary
Breaks the hydrogen strands apart
bonds between the Synthesizes daughter
two strands DNA strands
III
Alleviates
supercoiling Covalently links DNA
fragments together
Synthesizes an
RNA primer
Summery: Components of the replication apparatus
Catalyzed by
DNA topoisomerases
Termination of
Replication
8
DNA pol III makes only one mistake per 10 bases
made
There are several reasons why fidelity is high:
in the 5 to 3 direction
DNA Replication in Eukaryotes
Eukaryotic DNA Synthesis Is Similar to Synthesis in
Prokaryotes, but More Complex
In eukaryotic cells:
there is more DNA than prokaryotic cells
the chromosomes are linear & very long
the DNA is complexed with proteins
Contd .
Eukaryotic chromosomes contain multiple origins of
replication to allow the genome to be replicated in
timely manner.
DNA replication proceeds bi-directionally from many
origins of replication
Origin of replication in Eukaryotes Origin recognition complex
In eukaryotes, a hexametric
origin recognition complex
(ORC) binds to replication
origins and then recruit
additional factors (as Cdc6 and
Cdt1) that will themselves recruit
the hexameric MCM2-7 DNA
helicase to form a prereplicative
complex
This process occurs during
mitosis and along G1 and is
called DNA replication
licensing, a crucial regulation
of eukaryotic DNA replication
CDC6: cell division cycle
CDCT1: CDC10-dependent trans
this complex is still inactive, and only a subset of these reassembled
origins
will be activated in S phase
As the cells enter S Phase of the cell cycle DDK and CDK proteins
trigger initiation of replication.
DNA Pol/primase synthesizes primers
The primer: Template junction is recognized by sliding clamp
loader.
DNA pol and synthesize leading and lagging strands.
Contd
Eukaryotic DNA polymerases
Eukaryotic cells contain well over a dozen different
DNA polymerases(~15)
Four: alpha (), delta (d), epsilon (e) and gamma (g)
have the primary function of replicating DNA
, d and e Nuclear DNA
g Mitochondrial DNA
Other are involved in repair processes( next table )
Contd.
Eukaryotic replication
----parental DNA (with histones)
----Ribonulceases H1
DNA pol is the only polymerase to associate with
primase
The DNA pol /primase complex synthesizes a
nucleotides
This is used by DNA pol d or e for the processive
DNA pol d
The exchange of DNA pol for d or e is called a
polymerase switch
It occurs only after the RNA-DNA hybrid is made
DNA polymerases also play a role in DNA
repair
DNA pol b is not involved in DNA replication
It plays a role in base-excision repair
Removal of incorrect bases from damaged DNA
170