Molecular Biology ( 240386)

Prof. Raed Al-Atiyat

(Class material of Prof. Raida Khalil)

1st Sem. 2023-2024

@9.45 am Sat/Mon
Class Room # 2901
• Week 1

- Discuss course syllabus

- Introduction to course contents

- Introduction to DNA Structures and

other Macromolecules
 Introduction
• Foundational principles of cell biology : cells make up
a multicellular organism are autonomous!

• Proteins: majority of active cellular components

• Specific function of protein dictated by its sequence

amino acids, instructions encoded in sequence of
nucleotides in deoxyribonucleic acid (DNA),
biological information-carrying molecule.

• One copy of DNA of human genome encompassing

23 chromosomes & encodes 20,000 proteins.
• Human genome contains a total of base pairs ,
total
• information capacity bits,
corresponds to about
• bytes of information

• (unit used to measure computer memory).

• A single copy of the human genome weighs
Grams

• In theory, same information contained

• in a million one-terabyte laptop computer hard drives
stored in a mass of DNA the size of a grain of sand.
FIGURE 5-1 Overview of four basic molecular genetic processes
5.1 The Double-Helical Structure of DNA

• DNA and RNA have very similar primary structures

• DNA and RNA polymers in cells have quite different

three-dimensional structures

• Native DNA Is a Double Helix of

• Complementary Antiparallel Strands

• Purines: A and G pyrimidine : C; T and U

FIGURE 2-17 Chemical structures of the principal bases in
nucleic acids.
 consists of two phosphoester
bonds, one on the side of the
phosphate and another on the
side.

FIGURE 5-2 Chemical directionality of a nucleic acid strand

FIGURE 5-3 The DNA double helix.
• Almost all DNA in cells takes the form of a right-handed helix B
form DNA, normal form present in most DNA stretches in cells

• The x-ray diffraction pattern of DNA indicates stacked bases

regularly and spaced 0.34-nm apart along the helix axis.

• helix makes a complete turn every to , 10–10.5 base pairs

• outside of helix, spaces between intertwined strands form two

helical grooves of different widths, major groove and the minor
groove
• Atoms on edges of each base within these grooves accessible
from outside the helix, forming two types of binding surfaces:
• DNA-binding proteins can read sequence of bases in duplex DNA
by contacting atoms in either major or minor grooves.
In natural DNA, A hydrogen-bonds with T and G with C,

(a larger purine and a smaller pyrimidine)=

Watson-Crick base pairs.

complementary.
In theory and in synthetic DNAs, other base pairs form.
E.g: G hydrogen bonds with T within space available in
helix.
FIGURE 5-4
A- bidirectional pink arrow below chemical structures of
T-A and C-G indicates specific distance and angle
between bonds. T-A bond matches both distance and
angle of pink line and the C-G angle slightly steeper.

B- A bidirectional pink arrow below chemical structure

of C-A and T-G indicates the specific distance and angle
between bonds. Both C A and T G tilted to opposite
angle with the same distance
Any irregularities in DNA caused by chemical
damage or mispairing of bases will disrupt this
uniform structure and, will allow DNA repair
enzymes to identify and act on the site of damage.
• Important modifications in structure of B-form DNA
about as a result of protein binding to specific DNA
sequences.

• Multitude of hydrogen and hydrophobic bonds

between bases provides stability to DNA,

• Double helix flexible about its long axis Unlike the α

helix in proteins

Question : Explain why!

And importance of flexibility !!
FIGURE 3-4 The α helix, a common secondary structure in
proteins.
FIGURE 5-5 Interaction with a protein such as TBP can
bend DNA.
Why did DNA, rather than RNA, evolve to be the carrier of
genetic information in cells?
FIGURE 2-16 Common structure of nucleotides
FIGURE 5-6 Spontaneous hydrolysis of RNA catalyzed by the -hydroxyl
group. -hydroxyl group in RNA can act as a nucleophile, attacking
phosphodiester bond. cyclic monophosphate derivative further hydrolyzed
to a mixture of 2’ and 3’ monophosphates.
Week 2

The Structures and versatility of RNA

Chemical and Physical properties of Nucleic
Acids
DNA Can Undergo Reversible Strand
Separation
During replication and transcription of DNA
unwinding and separation of DNA strands=denaturation, or
melting : experimentally by increasing temperature of a solution
of DNA.
Mechanism
thermal energy increases: increase in molecular motion ;breaks
hydrogen bonds and other forces that stabilize double helix.
; strands separate, driven apart by electrostatic repulsion of
negatively charged deoxyribose-phosphate backbones of two
strands.

Near denaturation temperature, a small increase in temperature

causes loss of multiple weak interactions holding
strands together

.
 stacked base pairs in duplex DNA absorb less ultraviolet
(UV) light than unstacked bases in single-stranded DNA,
= hyperchromicity
EXPERIMENTAL FIGURE 5-7
Tm: melting temperature: DNA strands separate depends on
several factors:

• proportion of G·C pairs!

• Ion concentration of solution :negatively charged phosphate

groups two strands shielded by positively charged ions.

If ion concentration low! Consequences!

• Agents destabilize hydrogen bonds, e.g formamide or urea,

• Extreme PH: At low (acid) , bases become protonated;

positively charged, repelling each other.
At high (alkaline) ; bases lose protons and become
negatively charged, repelling each other
.
• Lowering temperature,
• increasing ion concentration, or neutralizing causes
two complementary strands to reassociate into a
perfect double helix= renaturation
Factors :
• Time ; DNA concentration; ion concentration.
• Two DNA strands not related in
sequence will remain as random coils and will not
renature,
but they will
not inhibit complementary DNA partner strands from
finding each other and renaturing.
Denaturation and renaturation of DNA are the basis of
nucleic acid hybridization!!!
 DNA Molecules Can Acquire
Torsional Stress
Many ----------------and ----------------!DNAs DNAs are circular
molecules. *(overwound or underwound,) explain!

Eukaryotic nuclear DNA -------------, long loops of DNA fixed

within -----------------!
torsional stress! Produced relieved by DNA supercoils

topoisomerase I vs topoisomerase,( function !)

Nick!

Refer to : KEY CONCEPTS OF SECTION 5.1

EXPERIMENTAL FIGURE 5-8 Topoisomerase I
relieves torsional stress on DNA
Week 3
The Replication of DNA
 5.2 DNA Replication

Definition : every cell division accompanied by exact copying

of DNA sequence of each chromosome
An understanding of biochemical mechanism for copying of
DNA came from isolation of first template-directed DNA
polymerase enzyme from extracts of Escherichia coli cells by
Arthur Kornberg and colleagues in 1956
DNA Polymerases Require a Template
and a Primer to Replicate DNA

DNA polymerases requirements for synthesis of a new

DNA strand:
i. A single-stranded DNA template.
ii. A DNA primer base paired with template, with a free ---
----------------at end
iii. deoxyribonucleoside -triphosphate (dNTPs) precursors.
 Description!! Read

FIGURE 5-9 DNA is synthesized from 5’ to 3 ‘dNTP

precursors.
DNA polymerases replicate DNA with extraordinary speed and
fidelity!!

• Add single nucleotide to growing chain in 2 millisec, not enough

time to pick up incorrectly bp nucleotide.

• higher degree of fidelity achieved by active site of polymerase

only accommodating base pairs with exact geometry of normal
Watson-Crick base pair while rejecting nonstandard base-pair
geometries
• error rates of about 1 incorrect nucleotide per (10,000)
polymerized nucleotides ( fig 5-4)
fidelity of DNA replication in most organisms about 1
mistake in 10 9(one billion) nucleotides incorporated into
a growing strand

WHY!
proofreading by DNA polymerases(. exonuclease activity of
3’ to 5’ direction)
FIGURE 5-10 Proofreading by DNA polymerase.
• All DNA polymerases have a similar 3D structure,

• “fingers” bind SS segment of template strand, and

polymerase catalytic activity (Pol) lies in junction between
fingers and “palm.”

• As long as correct nucleotides added to end of growing

strand 3’, remains in polymerase site.

• Incorporation of incorrect base at end 3’ ------ ----polymerase

pauses!
• primer strand not stably bp to template, move to
exonuclease site (Exo) where mispaired base removed , bp
properly with template and polymerization resume.
( C. M. Joyce and T. T. Steitz, 1995, J. Bacteriol. 177:6321, and S.
Bell and T. Baker, 1998, Cell 92:295)
Duplex DNA Unwound, and Daughter Strands Formed at DNA
Replication Fork

• two intertwined strands unwound(melted)--------Helicase ,

to make bases available for pairing with bases of dNTPs
(polymerized).

• Unwinding begins at replication origins (AT-rich); Vary

between organisms( Initiation step )

DNA polymerases cannot initiate replication de novo But

RNA polymerase can!! Explain
Priming :
RNA polymerase primase forms a short (∼12-nucleotide) RNA
primer complementary to unwound DNA template strands.
then elongated by DNA polymerase α for 25 nucleotides or so,
forming made of RNA at end 5’ and DNA at the 3’ end.
primer further extended by DNA polymerase δ, forming a new
daughter strand.

Replication fork !
Refer to description belong to this figure
 A DNA Replication Fork Advances by Cooperation
of Multiple Proteins

Model of Study Replication steps in eukaryotic

SV40 was the model !!
FIGURE 5-12 Model of an SV40 DNA
replication fork.
• large T-antigen, forms hexameric replicative helicase, uses
ATP hydrolysis to unwind parent strands at a replication
fork.

• Primers for leading and lagging daughter strands

synthesized by a complex of primase, (∼12 nucleotides),
and DNA polymerase α (Pol α), which extends RNA primer
with dNTPs for another 25 nucleotides (mixed RNA-DNA)

• short RNA-DNA primer extended by the high fidelity DNA

polymerase δ (Pol δ),(a proofreading mechanism 3’ to 5 ‘
based on exonuclease).
• During replication Pol δ synthesizes lagging-strand DNA, while
Pol ε, synthesizes most of length of leading strand.

• Pol δ and Pol ε each form a complex with PCNA(sliding

Clamp) which displaces the primase–Pol α complex following
primer synthesis

PCNA is a homotrimeric protein has a central hole through which

daughter duplex DNA passes, preventing PCNA–Pol δ and PCNA–Pol
ε complexes from dissociating from template.

pentameric protein : RFC (replication factor C; clamp loader ) : pen

PCNA ring it can encircle short region of double-stranded DNA
synthesized by Pol α.
 Parent DNA separated into ss templates at replication
fork, leading strand extended by Pol ε, which extend
growing strand up to replication fork.

SS template for lagging-strand synthesis bound by RPA, a

heterotrimeric protein ,maintains uniform conformation
template for copying by Pol δ.

Bound RPA proteins dislodged from parent strand by Pol

δ as it synthesizes complementary strand

topoisomerase I associates with parental ds DNA ahead of

to remove torsional stress introduced by unwinding of
parent strands
• Ribonuclease H and FEN I remove NTPs at ends of 5’ Okazaki
fragments; and replaced by dNTPs added by Pol δ as it
extends upstream Okazaki fragment.

• Okazaki fragments coupled by DNA ligase through standard

phosphodiester bonds.

• Refer to KEY CONCEPTS OF SECTION 5.2!

• DNA Replication Occurs Bidirectionally from Each Origin

• DNA replication from a single origin involve one

replication fork moves in one direction.

• two replication forks assemble at a single origin and move in

opposite directions, leading to bidirectional growth of both
daughter strands

▪ all bacterial, archaeal, and eukaryotic cells employ a

bidirectional mechanism of

▪ Read about SV40!

FIGURE 5-13 Bidirectional mechanism of DNA
replication
eukaryotic chromosomal DNA molecules contain
multiple replication origins

ORC, for origin recognition complex, binds to each

origin and associates with other proteins required to
load cellular hexameric helicases composed of six
homologous MCM (minichromosome maintenance)
proteins.

For more details refer to p875

Cell division begins with duplication of chromosomes;

initiation of DNA replication first step in cell division cycle.

Cellular DNA replication initiated by activation of MCM
helicase by protein kinase (DDK), which regulated in turn by S-
phase cyclin-dependent kinases.

Other cyclin-dependent kinases regulate mitosis and meiosis,

Week 4
DNA Repair and Recombination
 5.3 DNA Repair and Recombination
DNA damage caused by spontaneous cleavage of chemical
bonds in DNA, by environmental agents such as UV and by
reaction with genotoxic chemicals (by-products of normal
metabolism )or occur in environment.

mutation,(in germ cells vs somatic cells !! Consequently !

Chemical and Radiation Damage to
DNA Can Lead to Mutations
• DNA continually subjected to damaging ; estimates
number of DNA-damage a single human cell range from
104 to 106 to per day.

• Even DNA not exposed to damaging chemicals, aspects of

DNA structure inherently unstable.

e.g For example, bond connecting a purine to deoxyribose

prone to hydrolysis at a low rate under physiological
conditions, leaving a sugar without base------ coding
information lost ------mutation
• Normal cellular reactions, e.g movement of electron
along electron-transport chain in mitochondria and
lipid oxidation in peroxisome produce several
chemicals damage DNA, including hydroxyl radicals
and superoxide(O2-)-------mutation-----cancer

• Point mutation!explain !
• causes
• deamination of (C) base, converts into (U) base.

• deamination modified base 5-Methylcytosine into T

FIGURE 5-14 Deamination leads to point mutations
High-Fidelity DNA Excision-Repair Systems Recognize and
Repair Damage
• first elucidated through a combination of genetic and
biochemical studies in E. coli.
• error-free mechanisms arose early in evolution to protect
DNA
integrity.

• Mechanism : damaged DNA strand recognized, a segment of

damaged DNA strand excised, and gap filled by DNA
polymerase and ligase using complementary DNA strand as
template.
Base Excision Repairs T-G Mismatches and Damaged Bases
FIGURE 5-15 Base excision repair of a T·G mismatch. A
Figure 5.15
• A DNA glycosylase specific for G·T mismatches

• flips thymine base out of helix and then cuts it away from
sugar- phosphate DNA backbone (step 1 ), leaving
deoxyribose phosphate (black dot).

• An endonuclease : (apurinic endonuclease I, APE1) cuts

DNA backbone (step 2 ),
• deoxyribose phosphate removed by endonuclease,
apurinic lyase (AP lyase) associated with DNA polymerase β, a
specialized DNA polymerase used in repair (step 3 ).

• gap filled in by DNA Pol β and sealed by DNA ligase (step 4 ,

restoring original G·C base pair. See O. Schärer, 2003,
Angewandte Chemie 42:2946.
Mismatch Excision Repairs Other Mismatches and Small
Insertions and Deletions

Question : How the cell distinguish between newly

synthesized and old template DNA strands !!
FIGURE 5-16 Mismatch excision repair in human cells.
FIGURE 5-16 Mismatch excision repair in human cells. mismatch excision-
repair

• pathway corrects errors introduced during replication.

• A complex of MSH2 and MSH6 proteins (bacterial MutS homologs 1 and
6) binds to a mispaired segment of DNA (step 1 ).

• binding triggers binding of MLH1 and PMS2 (both homologs of bacterial

MutL).

• resulting DNA-protein complex then binds an endonuclease that cuts

newly synthesized daughter strand.

• DNA helicase unwinds helix, and exonuclease removes several

nucleotides from cut end of daughter strand, including mismatched
base (step 2 ).

• gap filled in by a DNA polymerase (Pol δ, in this case) and sealed by

DNA ligase (step 3 ).
Colon cancer and mutation( two copies) in repair
mismatch proteins( genes) ! , explain
Nucleotide Excision Repairs Chemical Adducts That
Distort Normal DNA Shape
FIGURE 5-17 Formation of thymine-thymine dimer
(a) thymine-thymine dimers caused by UV irradiation
(b) (b)These lesions repaired by excision-repair mechanism recognizes
distortion create in shape of the DNA double helix. red lines in (b)
represent UV-induced C — C bonds shown in (a).
FIGURE 5-18 Nucleotide excision repair in human cells.

• DNA lesion causes distortion of double helix, T-T dimers; recognized by

complex of XP-C (xeroderma pigmentosum C protein) and 23B proteins (step 1
).

• complex recruits transcription factor TFIIH ( helicase subunits and d by ATP

hydrolysis, unwind double helix.
• XP-G and RPA proteins bind to complex, unwind and stabilize helix by bubble
(about 25 bases) formed (step 2 )
• XP-G (endonuclease) and XP-F, (endonuclease), cut damaged strand at 24–32
bases apart on each side of lesion (releases DNA fragment as mononucleotides.
• gap filled by DNA polymerase , and nick sealed by DNA ligase

J. Hoeijmakers,2001, Nature 411:366, and O. Schärer, 2003, Angewandte Chemie

42:2946.

Question: find out the name of disesese resulted from mutation in

XP-G and other genes!
▪ If T-T dimer not in double helix, but in a ss DNA at a replication fork. Explain !

• progress of replication fork restored by ------------------!!!.

Ubiquitylation! PCNA! replacement of Pol δ or Pol ε ---------------, !Pol η. Features
!!

• features Pol η to form nonstandard base interactions,permitting replication

past T-T dimer on template,

• But polymerase making errors during replication of normal DNA.

• After replication has progressed past lesion, replication reset to the normal
replicative polymerase.

why mutagenesis with UV causes base changes in of T-T dinucleotides.?

Answer:
process of replication past a lesion in DNA ---- mutations caused by errors in
replication by translesion polymerase.
Two Systems Use Recombination to Repair Double-
Strand Breaks in DNA

1-Error-Prone Repair by Nonhomolohous End Joining

FIGURE 5-19 Nonhomologous end joining
• sister chromatids not available to help repair double-strand breaks, nucleotide
sequences butted together that not apposed in unbroken DNA.

• DNA ends from same chromosome locus and, when linked together, several
base pairs lost.

• Occasionally, ends from different chromosomes accidentally joined together.

• complex of, Ku and DNA-dependent protein kinase (DNA-PK), binds to ends of

a ds break

• . After formation of a synapse, ends processed by nucleases, resulting in

removal of a few bases and two ds molecules ligated together

• ds break repaired, but several base pairs at site of break removed.

See G. Chu, 1997, J. Biol. Chem. 272:24097; M. Lieber et

al., 1997, Curr. Opin. Genet. Devel. 7:99; and D. van Gant et al., 2001, Nat. Rev.
Genet. 2:196. (b) Structure of KU70/KU80 bound to a duplex DNA end. The
complex is shown
Repair of double-strand breaks by nonhomologous end joining pathway
can link segments of DNA from different chromosomes, forming an
oncogenic chromosomal translocation.
This repair mechanism also produces a small deletion, even when
segments from the same chromosome are joined.

Error-free repair of double-strand breaks in DNA is accomplished by

homologous recombination using undamaged sister chromatid as a
template.
Example of recombinational DNA repair is the repair of a
replication fork has come apart, known as a collapsed
replication fork.
 replication fork collapse

If a break in phosphodiester backbone (a nick) of one DNA strand not

repaired before a replication fork passes, replicated portions of daughter
chromosomes become separated when replication helicase reaches nick in
parent strand because there are no covalent bonds between two fragments of
parent strand on either side of nick.
FIGURE 5-20 Recombinational repair of a collapsed
replication fork.
2- Homologous Recombination Can Repair DNA Damage
and Generate Genetic Diversity

Read p 897 and 898!

BRCA1 or the BRCA2 gene and repair system----breast

cancer!

Repair of a Collapsed Replication Fork!( waiting students

to give short talk about not more 10 minutes!
FIGURE 5-20
 Repair of a Double-Strand Break by Homologous
Recombination
repair a ds break in chromosome and exchange large segments of
two ds DNA molecules (Figure 5-21)

• broken ends of DNA molecule digested by -5’ exonucleases, leaving ss region

of DNA with a 3’ end (step 1 ).

• RecA in bacteria or Rad51 in eukaryotes catalyzes invasion of one of these

ends into homologous region of homologous chromosome, (step 2 ).
• 3’ end of invading DNA strand extended by a DNA polymerase, displacing
parent strand as ss loop of DNA (dark blue) (step 3 ).
• loop extends to sequence complementary to other broken and -5’
exonuclease–digested end of DNA . complementary sequences base-pair
• end extended by a DNA polymerase using displaced ss loop of parent DNA
(dark blue) as a template (step 4 ).
• new ends ligated (step 5 ) to exonuclease-digested
Ends-----generates two Holliday structures in paired molecules (step
5 ).

• Branch migration of Holliday structures occur in either

direction (not diagrammed).

• Finally, cleavage of strands at positions shown by arrows, and ligation

of alternative and ends at each cleaved Holliday structure, generates
two recombinant chromosomes that contain DNA of one parent DNA
molecule on one side of initial break point (light and dark green
strands), and the DNA of other parent DNA on other side of break
point (light and dark blue) (step 6 ).

• region in immediate vicinity of initial break point forms a

heteroduplex, in which one strand from one parent base-paired to
complementary strand of other parent Bp mismatches between two
parent strands repaired by repair mechanisms to generate a
complementary bp
• In process, sequence differences between two parents lost,
(gene conversion).
FIGURE 5-21 Repair of a double-strand break
by homologous recombination
 FIGURE 5-22 Alternative resolution of a Holliday structure

Self reading ! KEY CONCEPTS OF SECTION 5.3