Nucleotide Metabolism

Biosynthesis and Degradation

Ezra Belay (M.Sc.)

Medical biochemistry Unit

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

Nucleoside = Nitrogenous base + ribose

Nucleotide = Nitrogenous base +ribose + phosphate

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

NMP cyclases &

*Lacks a 2-OH group phosphodiesterases
8
Structure of nucleotides

pyrimidine OR purine

N-b-glycosyl
bond

Ribose
or
2-deoxyribose

9
10
Biological functions of nucleotides

They are precursors of DNA and RNA

They are the energy currency in metabolic transactions.
They are components of:
Cofactors : such as NAD, FAD, S- adenosylmethionine,
and coenzyme A
Activated biosynthetic intermediates: such as UDP-
glucose and CDP-diacylglycerol.
Second messengers: such as cAMP and cGMP
Therapeutic targets: for cancer, bacterial infections.
Nucleotides and nucleosides also act as metabolic
allosteric regulators
E.g. ATP, AMP,ADP,NADH,NAD+ . 11
CO2 Asp

- 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 SAICAR Synthetase

H2N

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

Glycinamide Ribotide (GAR)

4 Formylglycinamide ribotide (FGAR) Inosine Monophosphate (IMP)
Sulfonamides

inhibit growth of bacteria

inhibit step 4 & 10
of purine biosynthesis

Humans unaffected by
sulfonamides.

They prevent the synthesis of folate by competing with its

p-aminobenzoate component
The antibiotic properties of sulfonamides are therefore
largely a result of their inhibition of nucleic acid 17
 Biosynthesis of AMP and GMP from IMP

Rxn8&9

***Amino group + energy

Mycophenolic acid (MPA)

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

Base specific nucleoside monophosphate kinases

Adenylate kinase kinase

AMP ADP ATP

ATP ADP ATP ADP

Nucleic acid synthesis
Guanylate kinase kinase
GMP GDP GTP

ATP ADP ATP ADP

Nucleoside monophosphate kinases Nucleoside diphosphate kinases

20
Regulation
ribose phosphate pyrophosphokinase

Gln:PRPP amidotransferase

Adenyosuuscinate synthase IMP dehydroginase

Purine nucleotide biosynthesis is regulated by feedback inhibition21

Purine Salvage pathway
In purine salvage pathway, purine bases formed by degradation of
RNA or DNA and intermediate of purine synthesis can be directly
converted to the corresponding nucleotides by salvage pathways
(~90%).
The significance of salvage pathway:
Save the fuel
Some tissues and organs such as brain and bone marrow are only
capable of synthesizing nucleotides by salvage pathway.
Two phosphoribosyl transferases are involved in purine salvage:
APRT (adenine phosphoribosyl transferase) for adenine.
HGPRT (hypoxanthine guanine phosphoribosyl transferase) for
guanine or hypoxanthine.
Both enzymes use PRPP as the source of ribose-5-phosphate
22
23

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

Inhibited by hydroxyurea and

8-hydroxyquinoline
Antibacterial and antitumor
agents

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

O PO3-2 Orotate Orotidine-5'-monophosphate

(OMP)
Reduced
Carbamoyl Phosphate Quinone OMP
Dihydroorotate Decarboxylase
Aspartate Dehydrogenase CO2
Aspartate Quinone O
Transcarbamoylase
(ATCase) O C
Pi
O
leflunomide HN CH

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

ATP ADP ATP ADP

(amination)

Azaserine (AS) & Acivicin are

Ammonia (in bacteria)
analogs of Gln.
inhibits the synthesis of CTP!
35
 Formation of dTMP
The immediate precursor of thymidylate (dTMP) is dUMP.
The formation of dUMP either by deamination of dCMP or by
hydrolyzation of dUDP.
dUTP
dUDP
dUTP diphosphohydrolase (dUTPase)
H2O
O MethylationO
Pi CH3
HN thymidylate synthase HN

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

uridine uridine-cytidine kinase UMP + ADP

cytidine + ATP
CMP
thymidine kinase
deoxythymidine + ATP dTMP + ADP

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

Out of body Xanthine

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

Degradation of pyrimidine nucleotides

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

Phosphate Sugars Purines

Bases Pyrimidines
(double ring)
group 5
HOCH2 OH
NH2 O (single ring)O
O N 6
CH3 H
4 4
1 7 5 1N 5 3N 5 3N
4
H H H 8 2
H H 9 4 6 2 6 2
3 1 1
3 2 N H N O H N O
N
O HO H H H H
D-Deoxyribose (in DNA) Adenine (A) Thymine (T) (in DNA) Uracil (U) (in RNA)
O P O

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

(a) Repeating unit of (b) Repeating unit of

deoxyribonucleic ribonucleic acid (RNA)
acid (DNA)

The structure of nucleotides found in (a) DNA

64
and (b) RNA
 DNA VS RNA

Deoxyribonucleotide

65
Ribonucleotide
Experimental works that Led to the Discovery of DNA
Structure

In 1953, James Watson and Francis Crick elucidated

the double helical structure of DNA
The scientific framework for their breakthrough was
provided by other scientists including
Linus Pauling
Rosalind Franklin and Maurice Wilkins
Erwin Chargaff

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

(a) An helix in a protein

67
Rosalind Franklin
She made marked advances in X-ray
diffraction techniques with DNA

The diffraction pattern she obtained

suggested several structural features of
DNA

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

There are three major

structural forms of DNA
depending on the
sequence as well as the
environment:
B form (Watson &Crick)
A form
Z form
 Central Dogma of Molecular Biology

Watson & crick immediately suggested a mechanism

for the transmission of genetic information.
They also suggested a means by which DNA could be
replicated.
 Chromosomes & Genomes
Genetic Terms
Chromosomes
complexes of DNA and proteins (RNAs) chromatin
Viral linear, circular; DNA or RNA
Bacteria single, circular
Eukaryotes multiple, linear
Genome
The genetic material that an organism possesses
Nuclear genome
Mitochondrial
Plasmid DNA
Genetic Terms.

Genes: A piece of DNA that encodes a particular trait

Locus: the location of a gene on a chromosome
allele: an alternate form of a gene
homozygous: individuals containing a pair of the same
alleles
heterozygous: individuasls containing two different alleles
for a gene
Dominant: expressed in the phenotype whether present as a
single or double copy
Recessive allele: not expressed because they are masked by
the dominant allele
genotype: the genetic composition of an individual
phenotype: the physical expression of a gene or allele
85
Viral Genomes
The genome can be
DNA or RNA
Single-stranded or double-stranded
Circular or linear
Viral genomes vary in size from a few thousand to more
than a hundred thousand nucleotides

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

The looped structure

compacts the
 Prokaryotic Gene (Operon) Structure
Stop Codon Stop Codon
+1 ATG TAA, TAG, TGA ATG TAA, TAG, TGA
Regulatory
DNA Elements Coding Sequence= ORF Coding Sequence= ORF

Promoter Protein A Protein B Terminator

& Operator sequence

Cistron 1 Cistron 2

Regulatory Sequences Structural or Coding Sequences

Regulatory and Coding Sequence Unit = Operon

Eukaryotic Chromosomes
Eukaryotic species contain one or more sets of
chromosomes
Each set is composed of several different linear
chromosomes
Chromosomes in eukaryotes are located in the nucleus
To fit in there, they must be highly compacted
This is accomplished by the binding of many proteins
The DNA-protein complex is termed chromatin
Eukaryotic genomes vary substantially in size
In many cases, this variation is not related to
complexity of the species
 Telomere

A eukaryotic chromosome contains a

long, linear DNA molecule Origin of replication
Three types of DNA sequences are
required for chromosomal replication
and segregation Origin of replication

Origins of replication Kinetochore proteins

Centromere
Centromeres
Telomeres Origin of replication

Genes are located between the

centromeric and telomeric regions
along the entire chromosome Origin of replication
A single chromosome usually has a
few hundred to several thousand Genes
Repetitive sequences
genes Telomere
 Eukaryotic Gene Structure

Start Codon Stop Codon

Cis- ATG TAA, TAG, TGA
Regulatory
Elements
Exon1 Exon2 Exon3
Promoter/
Enhancer
Eukaryotic genes are long & Have many introns
Exons (1.5%): encode for protein and RNA ( coding sequences)
introns(28.5%): noncoding sequences
Non-gene sequences:
Repetitive sequences (>50%) Tandem or interspersed
Moderately repetitive sequence: (150-300bp)
Found a few hundred to a few thousand times
Genes for rRNA and histones, Origins of replication & Transposons
Highly Repetitive DNA sequences (Satellite DNA)( up to 10 bp small)
Found tens of thousands to millions of times
E.g Alu family, genes in telomere & centromere
Regulatory sequences?
 Types of sequences in the human genome

Keys:

LINEs: Long interspersed elements

SINEs: Short interspersed elements
SSR: simple-sequence repeats
The Human Chromosomes
Normal human cells contain 23 pairs of
homologous chromosomes:
22 pairs of autosomes (autosomes: same in
both sex)
1 pair of sex chromosomes: XX in females &
XY in males.
One member of each chromosome pair is
derived from each parent.
Somatic cells have diploid complement of chromosomes i.e. 46
Germ cells (Gametes: sperm &ova)have haploid complement
i.e. 23
Individual chromosomes are recognized by
centromere position (metacentric, sub-metacentric,
acrocentric, telocentric)
arm lengths ( p- short, q -long)
 AutosomalPatterns of inheritance
recessive
dominant
Sex-linked traits
X-linked dominant
inheritance
X-linked recessive
inheritance
Y-linked inheritance
Mitochondrial inheritance
 Eukaryotic Chromatin Compaction
Stretched end to end, a single set of human chromosomes
will be about 2 meter long
nucleus is only 2 to 4 m in diameter

4
Therefore the DNA must be compacted ~10 -fold.

The compaction of linear DNA in eukaryotic

chromosomes involves interactions between DNA and
various proteins
Proteins bound to DNA are subject to change during the

life of the cell

These changes affect the degree of chromatin
compaction
Nucleosomes: The Basic Units of DNA Condensation
DNA is packaged by coiling around
a core of proteins known as histones.
The DNA-histone assembly is called a
nucleosome.
These are the repeating structural unit
within eukaryotic chromatin
The material of chromosomes, both
protein and DNA, is often referred to
as chromatin.
The protein component is about equal
in mass to the DNA component.
Histone proteins: the main packing PRTs

Basic proteins (rich in lysine &

arginine) that bind DNA
backbone
Play a role in the organization
and compaction of the
chromosome
Four core histones in
nucleosome
Two of each of H2A, H2B, H3 &
H4
Fifth histone, H1 is the linker
histone
 Nucleosomes Join to Form 30 nm Fiber
Nucleosomes
associate to form more
compact structure - the
30 nm fiber( solenoid
structure)

Histone H1 plays a role

in this compaction

A third level of compaction involves

interaction between the 30 nm fiber and the nuclear matrix
Radial loop domains
30 nm filaments is appear to be
organized in loops estimated at 40 to 100
kbp long.

The attachment of these loops to the

nuclear matrix(scaffold proteins) lead to
the formation of radial loop domains
these protein core then coils up
to further package the DNA into
the chromatids that are visible by
light microscopy in metaphase.
Chromatin
fibre loops
along the
protein
scaffold
Protein
More folds to make the most compacted Looped
structure chromatin fibre
folds

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. Linear eukaryotic replication

The linear eukaryotic
chromosomes contains far too
much DNA to be replicated
speedily from a single origin.
Eukaryotic replication initiates
at thousands of origins.
 Requirements of Replication
1. A template consisting of single-stranded DNA
2. Raw materials (substrates) to be assembled into a new
DNA
3. Many Enzymes/proteins
i. DNA Helicases : separates 2 strands
ii. SSBP: prevent reannealing of ssDNA
iii. DNA Topoisomerases: Prevents torsion by DNA
separation
iv. RNA Primase: RNA primer synthesis
v. DNA polymerase: synthesis of new strand
vi. DNA ligase: seals nick via phosphodiester linkage
 Features
Involves DNA templating
Starts at the specific origin
DNA replication is semiconservative
New duplex is parent new DNA

DNA replication is semi discontinuous

DNA replication may be uni/bi-directional
DNA replication requires RNA primer
RNA primer serve as a starter sequence for DNA
polymerase
Synthesis is always in the 5-3 direction
7 key issues that must be resolved during DNA replication:

unwinding of the helix

supercoiling
synthesis of a primer for initiation
discontinuous synthesis of the second strand
removal of the RNA primers
joining of fragments
proofreading
 DNA replication steps
1. Identification of the origin of replication
2. Initiation of DNA synthesis
Unwinding (denaturation) of dsDNA to ssDNA
template
Formation of the replication fork
3. Elongation
4. Termination of DNA replication
 Identification of origin of replication
DNA synthesis begins at a site termed the origin of
replication (oriC: origin of Chromosomal Replication)
Each bacterial chromosome has only one OriC
Three types of DNA sequences in oriC are functionally
significant
AT-rich region(13-bp)
DnaA boxes (5-TTATCCACA-3)
GATC methylation sites(11,
palindromic)
Contd.
DNA replication is initiated by
the binding of DnaA proteins to
the DnaA box sequences
This binding stimulates the
cooperative binding of an
additional 20 to 40 DnaA
proteins to form a large
complex
This causes the region to
wrap around the DnaA
proteins and separates the
AT-rich region
 Unwinding of dsDNA
DNA helicase separates the two DNA strands by breaking
the hydrogen bonds between them
This generates positive supercoiling ahead of each
replication fork
DNA gyrase travels ahead of the helicase and alleviates
these supercoils
Single-strand binding proteins bind to the separated DNA
strands to keep them apart
 Strand separation at the replication fork causes positive
supercoiling of the downstream double helix

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

DNA pol I and III

Normal replication

DNA pol II, IV and V

DNA repair and replication of damaged DNA

DNA polymerases I, II, and III possess 3' to 5'

exonuclease activity.
But only DNA polymerase I demonstrates 5' to 3'
exonuclease activity
DNA Polymerases.
DNA pol I
The first to be discovered

Composed of a single polypeptide with two subunits

o The smaller subunit 5 to 3 with exonuclease activity

o the large fragment or Klenow fragment, with

polymerization(5 to 3) and proofreading activities(3
to 5)
DNA pol I removes the RNA primers and fills the

resulting gap with deoxy nucleotides

After the gap is filled a covalent bond is still missing
DNA ligase catalyzes a phosphodiester bond
Thereby connecting the DNA fragments
 DNA Polymerases.
DNA pol III
Composed of 10 different subunits (Table next slide)
The subunit synthesizes DNA
The other 9 fulfill other functions
subunit is in the shape of a ring
It is termed the clamp protein
g subunit is needed for b to initially clamp onto the DNA
It is termed the clamp-loader protein
d, d and y subunits are needed for the optimal function of
the and b subunits
The complex of all 10 is referred to as the DNA pol III
holoenzyme
DNA Polymerase III.

DNA Polymerase III is a processive Enzyme

It remains attached to the template as it is synthesizing the daughter
strand
This processive feature is due to several different subunits in the
DNA pol III holoenzyme
In the absence of the subunit

DNA pol III falls off the DNA template after a few dozen

nucleotides have been polymerized

Its rate is ~ 20 nucleotides per second

In the presence of the subunit

DNA pol III stays on the DNA template long enough to

polymerize up to 50,000 nucleotides

Its rate is ~ 750 nucleotides per second
 DNA Polymerization (Elongation )
DNA polymerases can only synthesize DNA in the 5 to 3 direction
Therefore, the two new daughter strands are synthesized in different
ways
Leading strand

One RNA primer is made at the origin

DNA pol III attaches nucleotides in a 5 to 3 direction as it

slides toward the opening of the replication fork

Lagging strand

Synthesis is also in the 5 to 3 direction

However it occurs away from the replication fork

Many RNA primers are required

DNA pol III uses the RNA primers to synthesize small DNA

fragments (1000 to 2000 nucleotides each)

These are termed Okazaki fragments after their discoverers
 Contd.
As replication fork moves, only 1 strand can serve as
template for continuous DNA synthesisthe leading strand.
Opposite lagging strand undergoes discontinuous DNA
synthesis
The Reaction of DNA Polymerase
DNA polymerases catalyzes a phosphodiester bond between
the innermost phosphate group of the incoming
deoxynucleoside triphosphate & 3-OH of the sugar of the
previous deoxynucleotide
It requires a DNA template and all four dNTPs
DNA polymerases can elongate an existing DNA strand
(called a primer) but cannot initiate DNA synthesis.
Contd

The lagging strand is looped

This allows the attached DNA polymerase to synthesize the
Okazaki fragments in the normal 5 to 3 direction

-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

dnaA binds to origin DNA sequence

Primasome
dnaB helicase (unwinds DNA at origin)
dnaC binds dnaB
dnaG primase (synthesizes RNA primer)
DNA gyrase introduces negative supercoils ahead
of the replication fork
Rep protein helicase (unwinds DNA away from fork)
SSB binds to single-stranded DNA
DNA pol III primary replicating polymerase
DNA pol I removes primer and fills gap
DNA ligase seals gap by forming 3, 5-phosphodiester bond
Termination of Replication

Opposite to oriC is a pair of termination sequences called

ter sequences
These are designated T1 and T2

The protein tus (termination utilization substance) binds to

these sequences
It can then stop the movement of the replication forks

Finally DNA ligase covalently links all four DNA strands

Circular DNA replication often results in two intertwined
molecules
Intertwined circular molecules are termed catenanes

These are separated by the action of topoisomerases

Catenanes

Catalyzed by
DNA topoisomerases
 Termination of
Replication

Occurs @ specific site opposite ori c ~350 kb

Flanked by 6 nearly identical non- alindromic, 23
bp terminator (ter) sites
Proofreading Mechanisms
DNA replication exhibits a high degree of fidelity
Mistakes during the process are extremely rare

8
DNA pol III makes only one mistake per 10 bases

made
There are several reasons why fidelity is high:

1. Instability of mismatched pairs

Complementary base pairs have much higher stability

than mismatched pairs

This feature only accounts for part of the fidelity

It has an error rate of 1 per 1,000 nucleotides

Proofreading Mechanisms.

2. Configuration of the DNA polymerase active site

DNA polymerase is unlikely to catalyze bond
formation between mismatched pairs
This induced-fit phenomenon decreases the error rate

to a range of 1 in 100,000 to 1 million

3. Proofreading function of DNA polymerase
DNA polymerases can identify a mismatched
nucleotide and remove it from the daughter strand
The enzyme uses its 3 to 5 exonuclease activity to
remove the incorrect nucleotide
It then changes direction and resumes DNA synthesis

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)

----Polymerases (,d,e) Produce the

RNA/DNA chain
-------Pol initiation of replication (origins)
priming of Okazaki fragment
complex with DNA primase
(30 to 40 nucleotides)

-------Pol d synthesis of lagging strand

Pol e synthesis of leading strand

----accessories proteins: PCNA(~ =sliding clamp)
and Rf-C(~ clamp loader)

----Pol d,g, e have exonuclease activity ( 3to 5)

=proofreading

----Other Pols (pie, lambda, phi, rho, and mu)

do not have exonuclease activity ( 5to 3)
Proliferating Cell Nuclear Antigen: PCNA

----Ribonulceases H1
 DNA pol is the only polymerase to associate with
primase
The DNA pol /primase complex synthesizes a

short RNA-DNA hybrid

10 RNA nucleotides followed by 20 to 30 DNA

nucleotides
This is used by DNA pol d or e for the processive

elongation of the leading and lagging strands

Current evidence suggests a greater role for

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

Recently, more DNA polymerases have been

identified
Lesion-replicating polymerases
Involved in the replication of damaged DNA
They can synthesize a complementary strand over the
abnormal region
 Telomeres and DNA Replication
Linear eukaryotic chromosomes have telomeres at both ends
The term telomere refers to the complex of telomeric DNA
sequences and bound proteins
Telomeric sequences consist of
Moderately repetitive tandem arrays

3 overhang that is 12-16 nucleotides long

Telomeric sequences typically consist of Several

guanine nucleotides & often many thymine nucleotides
Telomeres..
but telomers are problematic to replicate
Lagging strand synthesis at end of chromosome is a
problem b/c once the RNA primer is removed, there is no
free 3'-hydroxyl group from which to elongate.
Telomerase directs synthesis of the telomere repeat
sequence to fill the gap.
This enzyme is a ribonucleoprotein w/an RNA that serves
as the template for the synthesis of its DNA complement.
Telomeres
Telomerase..
Telomerase highly active in >90% of tumors i.e. cell
immortalization and uncontrolled proliferation (drug
target)
~ 150 genes control telomere length in yeast and
shortening telomeres is associated with cell senescence
and death
Many adult cell types have detectable telomerase
activity, but it is a highly regulated, fine tuned activity
Nucleosomes and DNA Replication
Replication doubles the amount of DNA
Therefore the cell must synthesize more histones to
accommodate this increase

Synthesis of histones occurs during the S phase

Histones assemble into octamer structures
They associate with the newly made DNA very near the
replication fork

Thus following DNA replication, each daughter

strand has a mixture of old and new histones
Newly made
histone proteins
Thank you!!!
Blessed!

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