NUCLEOTIDES

STRUCTURE

 MINOR BASES (Modified, unusual)

Methylated bases
N6 methyladenine, N6,N7 methylguanine – mRNA
5-methylcytosine – DNA

 The nucleosides are coupled to D-ribose or 21 – deoxy-D-ribose thru a B-N-glycosidic bond

between the anomeric C of the ribose and the N9 of a purine or N1 of a pyrimidine
 Pseudouridine (C5-C’1 C’ glycosidic linkage) = tRNA
 Methylation by S-adenosylmethionine of a UMP of preformed tRNA forms TMP
 Alteration = after parent base has been incorporated into nucleic acid
 Direct measure of turnover of nucleic acid in cells
 In oligonucleotide recognition
 In regulating half life of RNAs

Free Heterocyclic Bases

Xanthine
Derivatives
Base + Ribose/deoxyRibose = Nucleoside
Nucleoside + Phosphate @ sugar = Nucleotide

BONDS
 for purines bond is at Nitrogen 9 of base and Carbon 1 of sugar
(n9-c1) Hydrogen
 for pymridines bond is at Nitrogen 1 and Carbon 1 of sugar bond
(n1-c1)

 for phosphate-ribose bond it is @ carbon 5 of ribose and oxygen of PO4

Ester Bond
PROPERTIES

TAUTOMERISM

 Anti B N-glycosidic configuration – Predominant tautomerism of bases

 Proper positioning of complementary bases in DNA
 Concentration of total Nucleotide= fixed with narrow limit (fixed inside the cell)
 Individual nucleotide varies depending on cell type and its energy state
*if the cell has high energy site  ATP
*if the cell has low energy site  ADP or AMP

Other properties:

 Strong absorption of UV light close to 260nm at pH 7 (conjugated double bonds)

 N glycosidic bond  stable to alkali (for both purines and pyrimidines)
In Pyrimidine  very stable to acid

In Purine  labile to acid

 Solubility
Nucleotides > Nucleosides > Free Bases

*Nucleotides have phosphate groups + OH groups

*Nucleosides have OH groups

POLYNUCLEOTIDES

 Formed by the condensation of two or more nucleotides.

 Condensation occurs between the 3’ -hydroxyl of one nucleotide and the OH of a 5’ -phosphate
of a second, with the elimination of H2O, forming a phosphodiester bond.
 Formation of bonds in nucleic acids exhibits directionality  proceeds in the 5’ 3’
 Write the nucleotide sequences from left to right synonymous with the 5’ -> 3’ direction as
shown: 5’-pGpApTpC-3’

NUCLEOTIDES: Chemistry and Metabolism

Structures of Nucleosides and Nucleotides

 A nucleoside is a base attached to ribose or deoxyribose
 A nucleotide is a nucleoside attached to one or more phosphate groups

Purine Base Nucleoside Nucleotide

(Base + (Nucleoside + Phosphate)
Pentose)
Adenine Adenosine Adenosine Monophosphate
(AMP)
Guanine Guanosine Guanosine Monophosphate
(GMP)
Pyrimidine
Base
Cytosine Cytidine Cytidine Monophosphate
(CMP)
Uracil Uridine Uridine Monophosphate
(UMP)
Thymine Thymidine Thymidine
Monophosphate (TMP)

 The nucleosides are coupled to D-ribose or 2’-deoxy-D-ribose through a -N-glycosidic bond

between the anomeric carbon of the ribose and the N9 of a purine or N1 of a pyrimidine
 There are two distinct orientations about the N-glycosidic bond: syn and anti conformations
(anti conformers predominate)

 Minor Bases (Modified, Unusual)

 Methylated bases
 N6 methyladenine and N6,N7 methylguanine  mRNA
 5-methylcytosine  DNA

 Pseudouridine (C5-C’1 C glycosidic linkage)  tRNA

 Arises by rearrangement of a UMP
 Uracil is attached by its carbon 5 to carbon ‘1 of D-ribose (carbon-carbon
glycosidic linkage) instead of a nitrogen-carbon glycosidic linkage
 Methylation by S-adenosylmethionine of a UMP of preformed tRNA forms TMP
 Alteration  after parent base has been incorporated into nucleic acid
 Other uses of Minor RNA
  Direct measure of turnover of nucleic acid in cells
 In oligonucleotide recognition
 In regulating half-life of RNAs

Derivatives = By products
By products: Xanthine and Uric acid
Differ only on the oxo group that is present in the ring
Hypoxanthine: 1 Oxo group (C6)
Xanthine: 2 Oxo Group (C2 & 6)
Uric Acid: 3 Oxo Group (C2, 6 & 8)

 Free Heterocyclic Bases Xanthine Derivatives

h
f

. E.g. Caffeine, a trimethylxanthine

Nucleotides: Properties
 Tautomerism
 Anti -N glycosidic configuration  predominant tautomerism of bases (oxo and amino)
 proper positioning of complementary bases in DNA
 Concentration of total nucleotide  fixed with narrow limit
 Individual NT varies depending on cell type and its energy state (Plenty of ATP);
Low energy state- ADP /AMP
  Strong absorption of UV light close to 260 nm at pH 7  due to conjugated double bonds
 N-glycosidic bond  stable to alkali (for both purines and pyrimidines)
 In pyrimidines, the bond is very stable to acid
 In purines, the bond is labile to acid
 Solubility: Nucleotides > Nucleosides > Free bases
o Nucleosides have pentose sugar (OH)
o Nucleotides are the most soluble because of the presence of Sugar and the phosphate
negative charges

Polynucleotides
 Polynucleotides are formed by the condensation of two or more nucleotides
 The condensation occurs between the 3’-hydroxyl of one nucleotide and the OH of a 5’-
phosphate of a second, with the elimination of H2O forming a phosphodiester bond
o (Condensation reaction with elimination of water formation of phosphodiester
linkages)
 The formation of bonds in nucleic acids exhibits directionality: proceeds in 5’  3’
 Write the nucleotide sequences from left to right synonymous with the 5’  3’ direction as
shown: 5’-pGpApTpC-3’
Nucleotides: Metabolic Roles
 Nucleotides are found primarily as the monomeric units of nucleic acids of cells
 DNA Precursors= dATP, dGTP, dCTP, TTP
 RNA Precursors = ATP, GTP, CTP, UTP
 Serve as energy stores for future use in phosphate transfer reactions  predominantly carried
out by ATP; GTP in protein synthesis (Transcription)
 Structural component of several coenzymes
 NAD+, NADP+, FAD (AMP)
 CoA (3’5’ ADP)
 Control numerous enzymatic reactions by being allosteric molecules in these reactions
 Serve as physiologic activators/mediators of numerous important cellular processes
 cAMP, cGMP  2nd messengers in signal transduction
 GTP-GDP  cascade events of signal transduction
 ADP  normal platelet aggregation
 Serve as activated intermediates in numerous biosynthetic reactions
 S-adenosylmethionine (SAM)
 “Activated” methionine in methyl transfer reactions
 As a source of propylamine in the synthesis of polyamines
 Sugar nucleotides
 UDP-Glc, Gal, GDP-Man, Fuc, CMP-sialic acid  glycogen and glycoprotein
synthesis
 CDP derivatives
 CDP-choline, ethanolamine, diacylglycerol  triglyceride and phospholipid
synthesis
 PAPS (3’ phosphoadenosine 5’ phosphosulfate)
 Sulfur transfer reactions for synthesis of glycosaminoglycans, glycoproteins,
sulfatides and conjugation reaction
- Cyclic GMP involve in the Visual(?) Cycle

 Serves as activated intermediates in numerous biosynthetic reaction

 CDP derivatives
 CDP choline, ethanolamine,diacylglycerol
 Triglyceride and phospholipid synthesis
 PAPS (3’phosphoadenosine 5’phophosulfate)
 S transfer reactions for the synthesis of Glycoaminoglycan,
glycoproteins, sulfatides and conjugation reaction
 UDP-glucuronide – conjugation reaction, bilirubin metabolism
 2 nucleotides that can play a role in Conjugation reaction are PAPS and UDP-glucuronide

Nucleotide Metabolism
 Dietary source  few or none are incorporated to tissue nucleotides
 Endogenous source
 De novo pathway
 Simple precursors
  Relatively high energy input
 Salvage pathway
 Free base  phosphoribosylation with PRPP
 Nucleoside  phosphorylation with ATP using a kinase
 Rates of synthesis  subject to precise regulation  nucleotides produced in appropriate
amount and at a time appropriate to meet cell needs
De Novo Pathway Salvage
Simple precursors Free base + Phosphate and ribose
Reaction: Phosphoribosylation
Source: PRPP
In Purine: Source of PRPP
(Ribose 6 phosphate + ATP
In Pyrimidines: source as PRPP
Relatively high Input energy Nucleoside
Reaction: Phosphorylation (always a
Kinase,
Source: ATP

 The overall determinant of the rate of de novo purine nucleotide biosynthesis is the
concentration of PRPP
 Origin of atoms in the purine ring system

 Multifunctional Catalysts
 Gene fusion ensures production of equal quantities of different catalytic activities
 Adjacent catalytic sites  facilitate rapid and complete transfer of intermediate
 Purine nucleotide de novo pathway:
1. +Glycine, +N5,N10-methenyl-H4 folate, ring closure (3, 4, and 6)
2. +Carbon dioxide, +Aspartate (7, 8)
3. +N10-Formyl-H4 folate, ring closure (10, 11)
 Regulation of Purine Nucleotide Synthesis
 Pyrimidine Nucleotide De Novo Pathway
 CAD (Carbamoyl phosphate synthetase II, Aspartate transcarbamoylase,
Dihydroorotase)
 UMP Synthase (Orotate phosphoribosyl-transferase, OMP decarboxylase)
 Regulation of Pyrimidine Nucleotide Synthesis

 Origin of atoms in the pyrimidine ring

 Salvage Pathways
  Purine Nucleotide Cycle
 Net effect: Deamination of aspartate to fumarate
 The cycle is very important in muscle cells  muscle cells lack most of the enzymes of
major anaplerotic reactions

 Formation of Deoxyribonucleotides
 dATP  inhibitor of all ribonucleotide reductases

 Purine nucleotide degradation

 Adenosine deaminase deficiency  combined immunodeficiency
 Purine nucleotide phosphorylase deficiency  T cell immunodeficiency

Synthetic Nucleotide Analogs

 Nucleotide analogs  chemically synthesized
 Inhibit specific enzymatic activities involved in synthesis of nucleotides and DNA
 Disrupt normal replication by interfering with the formation of W-C base pairing
 Synthesis of DNA  kill rapidly dividing cells (tumor cells and viruses)  anti-tumor agents and
antiviral agents
 E.g. 6-mercaptopurine, 5-fluorouracil, 5-iodo-2’-deoxyuridine, and 6-thioguanine
 Compounds that interfere with nucleotide metabolism
 Glutamine Antagonists
 Inhibit amidation reactions
 Azaserine and Diazonorleucine
 Antifolate
 Inhibits dihydrofolate reductase
 Methotrexate, Trimethoprim (Antibiotic)
 Antimetabolites
 Structural analogs of nucleotides which interfere with specific metabolic site
  6-mercaptopurine  as nucleotide  negative effector to PRPP Glutamyl Amidotransferase,
inhibits IMP Dehydrogenase, and Adenylosuccinase
 Has therapeutic potential
 As antiviral agents  used to interfere with the replication of viruses
 AZT (3’-azido-2’,3’-dideoxythymidine)  as 5’ triphosphate  inhibits reverse
transcriptase
 ddI (2’,3’-dideoxyinosine) and ddC  as triphosphate  becomes incorporated
into viral DNA but blocks further replication chain elongation because of
absence of 3’ OH terminus
 Adenine arabinoside  as AraATP  inhibits DNA polymerase
 Cytosine arabinoside  as AraCTP  competitive inhibitor with respect to dCTP
for DNA polymerase
 5-fluorouracil  FUTP  inhibits maturation of 45s rRNA
 Purine analogs are used to treat gout
 Used to inhibit xanthine oxidase activity
 Most common is allopurinol
 Several nucleotide analogs suppress immune response after organ transplantation to
reduce the likelihood of transplant rejection
 Azathioprine  6-mercaptopurine
URIC ACID
Normal adult humans
Synthesize between 600 and 700 mg/day
Dietary purines contribute = 300 to 600 mg
Total miscible pool: 1200 mg
Turnover at steady state – 600 mg/24 hours
Serum levels below 7 mg/100 ml is normal
Hyperuricemia (> 7mg/100 ml) may lead to gout
SSXs – Acute gouty arthritis
Tophi
Uric acid nephrolithiasis

URIC ACID SECRETION

The end product of purine catabolism is uric acid which must be excreted. Excretion occurs
via the kidney and gastrointestinal tract.
Adenine and Guanine

Moieties of Dietary Renal: 1/2

Nucleoproteins and
Nucleotides

Miscible pool
INPUT of Uric acid OUTPUT

Adenine and Guanosine

Extrarenal: ½
Moieties of Dietary
Into Gastrointestinal tract
Nucleoproteins and
Undergoes Bacterial
Nucleotides
degradation

1. Complete Glomerular filtration

2. Active Tubular reabsorption
3. Active Tubular Secretion

HYPERURICEMIA
- An overproduction of uric acid or
- A reduced rate of renal excretion
METABOLIC HYPERURICEMIA
PRIMARY
SECONDARY
RENAL HYPERURICEMIA

 METABOLIC HYPERICEMIA
Overproduction of uric acid may be due to
PRIMARY
- Enzymatic abnormality involving/affecting purine synthesis --- Increased
degradation
- The rate of the de novo biosynthesis of purines is determined by the availability
of PRPP.
SECONDARY
- An increased turnover of nucleic acids
- May be observed in leukemia, polycythemia vera, sickle cell anemia, and
psoriasis.

PRIMARY METABOLIC HYPERURICEMIA

DISORDER DEFECT NATURE OF DEFECT

Gout PRPP Synthetase Increased enzyme activity
due to elevated Vmax
Gout PRPP Synthetase Enzyme is resistant to
feed-back inhibition
Gout PRPP Synthetase Enzyme has increased
affinity for ribose-5-
phosphate (lowered Km)
Gout HGPRTase Partially defective enzyme
Von Gierke’s Disease Glucose-6-Phosphatase Enzyme deficiency

RENAL HYPERURICEMIA
A reduced rate of renal excretion
Thiazides- increased tubular reabsorption
ASA, Pyrazinoic acid – competes for secretion
Organic acids, such as lactate or ketone bodies, interferes with urate secretion -> DM, Glycogen
storage Disease 1, Alcohol intoxication, and Starvation -> Hyperuricemia
In chronic renal failure, the capacity of the kidney to secrete uric acid is lost more quickly than
the reabsorption capability -> hyperuricemia
METABOLIC DISORDERS
LESCH-NYHAN SYNDROME
Results the loss of functional HGPRTase gene -> complete lack of enzyme
A sex linked trait
Exhibit severe symptoms of gout; severe malfunction of the nervous symptom -> self
mutilation, mental retardation.
Death usually occurs before patients reach their 20th year
HYPOURICEMIA – Xanthine oxidase deficiency
<- Genetic or sever liver damage
  Xanthuria, Xanthine lithiasis
OROTIC ACIDURIA
I = deficiency of OPRTase and orotate decarboxylase
II = deficiency of orotate decarboxylase

 Seen in Reye syndrome = severely damaged mitochondria inability to utilize carbamoyl

phosphate
 Deficiency or Urea cycle enzyme (Ornithine transcarbamoylase)
 Allopurinol intake (affects UMP synthase)

Competes for OPRTase

Product formed inhibits OMP decarboxylase

MOLECULAR GENETICS

How genetic information is transmitted and expressed on the molecular levels

Nucleic Acids
 Macromolecular structures storage and expression of all the genetic information necessary for
building and maintaining life
 DNA (Deoxyribonucleic Acid) ® considered as repository of the genetic information; chemical
basis of heredity
 RNAs (Ribonucleic Acids) ® regarded as vectors and translators of the information; selective
expression of genetic information
 Polynucleotides whose phosphate bridges 3’ and 5’ positions of successive sugar residues
 Phosphodiester groups are acidic at pH 7.4 ® polyanions
 Bases (thymine – tRNA)
 Number of strand
 Chargaff’s rule
 Alkali treatment ® 2’, 3’ cyclic diester of mononucleotide (RNA)

DNA
 2 unbranched polynucleotide strands held together in an antiparallel manner
 Phosphate backbone is always located outside
 Bases are well stacked in interior
 Interwinding ® structure with helical grooves
 Complementarity
 Geometric factors
 Restriction imposed by phosphodiester bonds
 Anti glycosidic bonds
 Electronic specificity of interaction between complementary bases ® predominant
tautomers
 Forces that Stabilize the DNA Structure
 Mainly stabilized by hydrogen bonds between base pairs
  Stacking interaction of the bases inside
 Hydrophobic interactions of the bases
 Van der Waals forces
 Forms of DNA
 Sequence content, Ionic conditions, Degree of hydration ® B and Z DNA

B DNA Z DNA
Sequence --- pCpG nucleotide
content
Ionic Low ionic ---
conditions strength
Degree of High degree of ---
hydration hydration
Handedness Right Left
Base pairs/turn Ten Twelve
Helix rise/base 3.4 Å 3.7 Å
pair
Helix pitch 34 Å 45 Å
Groove Major and Single
Minor grooves
Forces Hydrogen Hydrogen bonds, Stacking,
bonds, Stacking, Hydrophobic, Van der
Hydrophobic, Waals, High
Van der Waals salt/polyamines, Binding to
specific protein,
CH3dcytidine(methyldeoxy
cytidine)
Other Most common Dispersed, Exerts
characteristics regulatory effect

 Thermal Properties of DNA

 In vitro, high temperature ® the hydrogen bonds between bases become unstable and
the strands of the helix separate in a process of thermal denaturation
 Regions of the duplex that have predominantly A-T base pairs will be less thermally
stable than those rich in G-C base pairs
 A-T base pairs are linked with two hydrogen bonds only while G-C base pairs are
linked with three hydrogen bonds
 In thermal denaturation, a point is reached at which 50% of the DNA molecule exists as
single strands ® melting temperature Tm
 When thermally melted ssDNA are cooled, the complementary strands will again re-
form the correct base pairs, in a process termed annealing or hybridization

Mitochondrial DNA
 Is circular, double-stranded, and composed of heavy (H) and light (L) chains or strands
 Contains 16,569 base pairs
 Encodes 13 protein subunits of the respiratory chain (of a total of about 67)
 7 subunits of NADH dehydrogenase (complex I)
  Cytochrome b of complex III
 3 subunits of cytochrome oxidase (complex IV)
 2 subunits of ATP synthase
 Encodes large (16S) and small (12S) mt ribosomal RNAs
 Encodes 22 mt tRNA molecules
 Genetic code differs slightly from the standard code
 UGA (standard stop codon) is read as Tryptophan
 AGA and AGG (standard codons for Arginine) are read as stop codons
 Contains very few untranslated sequences
 High mutation rate (5-10 times that of nuclear DNA)
 Comparisons of mt DNA sequences provide evidence about evolutionary origins of primates and
other species

RNAs
 Messenger RNAs (mRNAs)

 This class of RNAs are the genetic coding templates

used by the translational machinery to determine
the order of amino acids incorporated into an
elongating polypeptide in the process of translation

 2 – 5%, unstable to very stable, most heterogeneous

in size, 75 to 3000 NTs

 5’ capped by 7-methyl GTP

 Positive (+) effect on initiation of message
translation
 Protective role

 3’ Poly (A) tail

 Protective role
 Transport
 Interacting with ribosomes

 6-methyladenylates and 2’-O-ribose-methylated

nucleotides

 Transfer RNAs (tRNAs)

 This class of small RNAs form covalent attachments to

individual amino acids and recognize the encoded
 sequences of the mRNAs to allow correct insertion of amino acids into the
elongating polypeptide chain

 10 – 20%, 75 to 90 nucleotides, less stable

 Assume secondary structure  cloverleaf (4 arms)

 Acceptor arm = site of attachment of specific amino acid
 T C arm = binding of amino acyl-tRNA to ribosomal surface
 D arm = proper recognition of a given tRNA to its amino acid-tRNA
synthetase
 Anti-codon arm =
recognizes the triplet
codon on mRNA

 10% unusual bases

 Ribosomal RNAs (rRNAs)

 This class of RNAs are assembled,

together with numerous ribosomal proteins, to form the ribosomes
 80S Ribosome divided into two subunits: 60S and 40S

 60S Ribosomal subunit

 Has peptidyl
transferase activity

 28S rRNA, 5.8S rRNA,

5S rRNA

 49 ribosomal
proteins

 40S Ribosomal subunit

 18S rRNA

 33 ribosomal
proteins

 80%, 100 to 4,700 nucleotides

  Peptidyl transferase activity

 Highly methylated

 Small RNAs

 Small nuclear RNAs, SNURPs, snRNA

 90 to 300 nucleotides
 Roles

 Cellular architecture

 Gene regulation

 Transport of RNAs

 RNA processing

 Splicing -
endonucleolytic
cleavage and
ligation

 Poly A addition

 Large and small noncoding regulatory

RNAs

 miRNAs and siRNAs

 Inhibition of gene expression

 20 – 22 nucleotides in length (21 – 25 nucleotides)

 Exciting new potential targets for drug development

 Micro-RNAs (miRNAs)

 Nucleolytic processing of products of distinct gene

 Imperfect RNA-RNA hybrid

  Translational repression, mRNA destabilization, and mRNA
degradation
 Silencing RNAs (siRNAs)

 Nucleolytic processing of large dsRNAs ® endogenous/viral

 Perfect RNA-RNA hybrid

 Induce RNA cleavage ® mRNA inactivation

 Long noncoding RNAs (lncRNAs)

 Do not code for protein; range in size from 300 to 1000s of

nucleotides in length

 > 90% of all eukaryotic genomic DNA is transcribed ® ncRNAs

make up significant portion of this

 Roles

 Structural - chromatin

 Regulatory - mRNA gene transcription by RNA polymerase

II

Chromatin
 In eukaryotic cells, DNA is packaged in chromatin
 Chromatin ® DNA, Histones, Nonhistonic, acidic, larger proteins, RNA
 Histone proteins (H1, H2A, H2B, H3, and H4) ® globular basic proteins rich in arginine and lysine
at N ends ® polycationic
 The binding of DNA by the histones generates a structure called the nucleosome ® periodic
structure (“beads on a string”)
 Nucleosome core ® an octamer ® (H2A-H2B)2 dimer, (H3)2 (H4) tetramer
 H1 occupies the internucleosomal DNA histone ® linker histone
  The DNA helix (146 base pairs) coils twice (1.75) around the nucleosome core/histone octamer
through electrostatic interactions between the negatively charged phosphate groups in
nucleotides and the positively charged basic amino acids in histones
 The linker DNA segments between each nucleosome can vary from 20 to more than 200 base
pairs
 Nucleoplasmin
 Acts as “molecular chaperone”
 Non-random distribution called phasing
 Modifications of chromosome structure and their effects
 Acetylation
 H3 and H4 ® regulates transcription
 Core histones ® chromosome assembly during replication
 Phosphorylation
 H1 ® condensation of chromosomes during replication cycle
 ADP Ribosylation ® DNA repair
 Methylation ® regulates transcription
 Monoubiquitylation ® associated with gene activation, repression
 Sumoylation ® transcription, repression
 The Packing or Compaction Ratios of Each of the Orders of DNA Structure

Chromatin Form Packing

Ratio
Naked double-helical DNA ~1.0
10-nm fibril of nucleosomes 7 – 10
30-nm chromatin fiber of superhelical 40 – 60
nucleosomes
Condensed metaphase chromosome 8000
loops

 The protein-DNA structure of chromatin is stabilized by attachment to a non-histone protein

scaffold called the nuclear matrix

Chromosome
 Between cell divisions (Interphase) ® chromatin exists as a tangle of fibers of 10 – 30 nm (10-
9m) diameter and 0.25 – 2 mm length
 Euchromatin
 Heterochromatin ® constitutive or facultative
 Just before a cell division (Mitosis), the chromatin condenses into metaphase chromosomes
 Two-fold symmetry ® two identical, symmetrical DNA molecules called chromatids
 The chromatids are joined by a centromere (130 AT base pairs)
 Kinetochore (centromere + proteins) ® provides anchor for mitotic spindle ®
chromosomal segregation during metaphase
  Telomeres (short TG-rich repeats) ® shortening is associated with malignant
transformation and aging

Mammalian Genomes
 Haploid genome ® 3.5 x 109 base pairs ® 100,000 essential proteins
 Presence of a large amount of non-coding sequences
 Functions
 Some sequences are involved in the control of gene expression during development,
differentiation, and adaptation to environment
 Act as an evolutionary buffer able to withstand nucleotide mutation without disrupting
the integrity of the organism ® permit genetic rearrangement by allowing
recombination to occur more rapidly

Sequence Arrangement in Eukaryotic DNA

I. More than half of the DNA in Eukaryotic Organisms is in Unique or Nonrepetitive Sequences.

II. In human DNA, at least 30% of the Genome consists of Repetitive Sequences.

BROAD CLASSIFICATION OF REPETITIVE SEQUENCE DNA:

A. Moderately Repetitive
- contains about 50 times repetition of sequences
- these are not clustered but are interspersed with unique sequences
- Classified as:
o Long Interspersed Repeat Sequences (LINEs)
 This is more prone to mutation because they can’t be regulated
efficiently due to their length
o Short Interspersed Repeat Sequences (SINEs)
 Contains the Alu B1 and Alu B2 family
 Present in about 500,000 copies per haploid genome
 Accounts for ~10% of the human genome
 The Alu family of SINEs are transcribed as integral
components of mRNA precursors or as discrete RNA
molecules
 They regulate mRNA transcription and splicing
 Members of the SINEs may be mobiles elements: capable of
jumping into and out of various sites within the genome

o LINEs and SINEs are Retroposons: they are DNA fragments that arose from
movement from one location to another in the chromosome through an
RNA intermediate, by the action of reverse transcriptase that trancribes
DNA sequences from RNA.

B. Highly Repetitive
- Consists of 5 to 500 base pairs that are repeated in tandem
 - They are often clustered in centromeres and telomeres of the chromosome
- The majority are transcriptionally inactive
- They play a structural role

III. Microsatellite Repeat Sequences

- exists both in Dispersed or Group Tandem Arrays
- the sequences contains 2 to 6 base pairs repeated up to 50 times

- commonly found as dinucleotide repeats of:

AC on one strand: occur at 50,000 – 100,000 locations in the genome
TG on the other strand

- CG, AT, and CA can also occur at times

- a heritable trait

- they are useful in constructing genetic linkage maps because they are easiy detected using
Polymerase Chain Reaction (PCR).
: most genes have microsatellite markers so when there is mutation or abnomality in a
certain microsatellite (microsatellite polymorphism), PCR can be used to detect the
gene mutation that has caused a certain disease

DNA REPLICATION
Function : provision of progeny with genetic information of parent cell
• Occurs with high fidelity → maintain genetic stability within organism & species
• Semi-conservative process
• Complex, highly coordinated series of reactions
- both strands serve as templates
- multifocal, bidrectional (50 NT vs. 500 NT/sec)
- 5’ → 3’ direction
➡Continuous : Leading strand
➡Discontinuous : Lagging strand
- requires primer (short RNA stretch : 10 NT)
1. Identification of the origins of replication
- O protein interacts with Ori element
2. Local unwinding of AT rich region → ssDNA → template
3. Replication fork
4. Initiation & elongation
5. Replication bubbles → ligation
6. Reconstitution of chromatin structure
• SSB proteins - prevent premature re-annealing of dsDNA
• Helicase - processive unwinding
• Primase - initiates synthesis of RNA primers
• Topoisomerases - relieve torsional strain
• DNA polymerase III - dexoynucleotide polymerization
• DNA ligase - seals ss nick between nascent
chain & Okazaki fragments

Synthesis of DNA proceeds in the 5’ → 3’ direction

• Processivity - ability of a particular polymerase to remain associated with the template strand
• The longer it associates, the higher the processivity of enzyme
- DNA polymerase processivity is enhanced by additional protein activites of the replisome,
identified as processivity accesory proteins
 DNA pol δ

DNA pol ε

Prokaryotic DNA polymerase Eukaryotic DNA polymerase

DNA polymerase III • DNA pol ε (epsilon) - processive, leading strand

5’ → 3’ polymerase • DNA pol δ (delta) - processive, lagging strand
3’ → 5’ exonuclease (proofreading/editing)
5’ → 3’ exonuclease

DNA polymerase I • DNA pol α (alpha) - primase

- repair of damaged DNA • DNA pol β (beta) - DNA repair
DNA pol Ɣ (gamma) - mitochondrial replication