Medical Biology and Genetics
for Pharmacy
2020-2021
Lecture 4: DNA replication and repair
mechanisms
Assist. Prof. Berrak ÇAĞLAYAN bcaglayan@[Link]
Learning Objectives
✓ To understand the historical theories of DNA replication.
✓ To outline the steps of DNA replication.
✓ To identify the enzymes involved in replication and define their functions.
✓ To identify the key proofreading processes in DNA replication.
✓ To describe the role of telomeres.
✓ To compare the roles played by centromeres, telomeres, and replication
origins.
✓ To link the DNA damage factors, mutations and repair mechanisms.
✓ To identify the major types of DNA mutations.
✓ To describe DNA damage repair mechanisms and associated diseases.
✓ To understand advanced DNA repair mechanisms and their clinical uses.
Three models were proposed for DNA replication
Meselson and Stahl usedE. coli grown in heavy nitrogen (15N) to
understandDNA replication.
Meselson-Stahl
Experiment
Video
After one round of cell division the DNA sediments were halfway
between the 15N and 14N levels, indicating that it contains half of
each. In subsequent cell divisions, an increasing amount of DNA
contains 14N.
DNA replication is semiconservative, meaning that
half of the original molecule (one of the two
strands in the double helix) is “conserved” in the
new molecule.
In each round of DNA replication, each of the two
strands of DNA is used as a template for the formation
of a new, complementary strand.
The original strand is
referred to as the
template strand
because it provides the
information, or the
template, for the newly
synthesized strand.
DNA synthesis begins at
replication origins
DNA synthesis occurs at replication forks
The two replication forks move away in opposite
directions at each replication origin
A new DNA strand is synthesized in the5’ -> 3’
direction
DNA polymerase catalyzes DNA synthesis
DNA polymerase contains separate sites for DNA
synthesisand proofreading (self correcting)
During DNA synthesis,
DNA polymerase
proofreads its own
work
At a replication fork, the two newly synthesized
DNA strands are of opposite polarities
At each replication fork, the lagging DNA strand is
synthesized in pieces
DNA
replication
Video
At each replication fork, the lagging DNA strandis
synthesized in pieces
Leading strand is synthesized continuously
Lagging strand is synthesized discontinuously
Short RNA primers are synthesized by primase
(RNA polymerase) on the lagging strand for DNA
synthesis
Multiple enzymes are
required to synthesize
Okazaki fragments on
the lagging strand.
RNA primers are erased
and replaced with DNA
in order to produce a
continuous DNA chain
from Okazaki fragments
at the lagging strand.
DNA ligase joins together Okazaki fragments on
the laggingstrand to complete the process
Special proteins help to open up the DNA double
helix in front of the replication fork
DNA helicases hydrolyze ATP to open up the DNA
double helix in front of the replication fork
DNA synthesis is carried out by a group of proteins
that act together as a replication machine
DNA replication
animation
DNA topoisomerases relieve the tension that
builds up in front of a replication fork
DNA would be lost during each round of cell
division without a special mechanism to replicate
the ends of chromosomes قد يضيع الحمض النووي خالل كل جولة من انقسام
الخاليا دون آلية خاصة لتكرار نهايات الكروموسومات
Telomeres are packaged into specialized
structures (t-loops) that protect the ends of
chromosomes ) التي تحميt-loops( يتم حزم التيلوميرات في الهياكل المتخصصة
نهايات الكروموسومات
Telomeres replicates the ends of
eukaryoticchromosomes. Telomeres and
telomeraseprevent linear eukaryotic chromosomes
from shortening with each cell division.
Telomerase animation
Telomerelengthis regulatedby cellsandorganisms
Bacterial chromosomes typically havea singleoriginof
replication
Bacterial replicationoriginis temporarily blockedafter the
replicationis startedto prevent theinitiationof
morethanone roundof replicationduringa
singlecelldivision.
InhibitingDNA replicationcan be helpful in treatment of
hyperproliferativediseases
Purine and pyrimidine nucleoside («Trojan» or «suicide») analogs can directly
inhibit DNA polymerase activity after being incorporated into DNA.
FDA approvednucleosideanalogsarethe largest class of
antineoplastic agents used clinically (~20% of all drugs
used in chemotherapy)
DNA damagingagentsarealsousedin cancer becauseof
their abilityto inhibit DNA polymerase
DNA damaging agents react with nucleotides and modify the structure of DNA.
The modified structure then acts as a physical barrier and hinders the movement
of DNA polymerase.
Inhibitorsof bacterialDNA replicationareusedas antibiotics
Aminocoumarins and quinolones are used for treatment of Gram- and Gram+
bacterial infections.
They inhibit DNA gyrase (DNA topoisomerase II) and DNA topoisomerase IV and
block DNA synthesis in bacteria.
Effects on mtDNA?
DNA
Repair
DNA repair is required to correct DNA damage
DNA damage can occur anytime during cell cycle due
to:
• Heat
• Metabolic accidents
• Radiation
• Exposure to DNA damaging agents (free radicals,
reactive oxygen-oxidative stress…)
Failure to repair DNA damage can have severe
consequences for a cell or organism
Cancer incidence
increases
exponentially with
age!
Errors made during
DNA replication
mustbe corrected to
avoid mutations
Whenever the replication machinery
makes a copying mistake, it leaves
behind a mispaired nucleotide
(commonly called a mismatch). If
left uncorrected, the mismatch will
result in a permanent mutation in the next round of DNA replication.
WithoutDNA repair, spontaneous DNA damage
would rapidly change DNA sequences
A single nucleotide changecauses the disease
sicklecell anemia
Depurination and deaminationare the most
frequent chemical reactions knownto create
serious DNA damage in cells
Depurination produces mutations(if left
unrepaired)
Deaminationproduces mutations (if left
unrepaired)
UV radiation in sunlight can cause the formationof
thyminedimers
Covalent linkage between thymine bases may
result in the dimer being replicated as a single
base, which results in a frameshift mutation
DNA conformation is searched for unusual bases by
special enzymes that travel along DNA.
The basic mechanism of DNA repair involves three steps:
Step 1 (excision): the damage is cut out
by one of a series of nucleases, each
specialized for a type of DNA damage.
Step 2 (resynthesis): the original DNA
sequence is restored by a repair DNA
polymerase, which fills in the gap created
by the excision events.
Step 3 (ligation): DNA ligase seals the nick
left in the sugar–phosphate backbone of
the repaired strand. Nick sealing, which
requires energy from ATP
hydrolysis,remakes the broken
phosphodiester bond between the
adjacent nucleotides.
Xeroderma pigmentosum (XP) is an inherited
(autosomal recessive) condition characterized by an
extreme sensitivity to ultraviolet (UV) rays from
sunlight
Mutations in the nucleotide excision repair enzymes which repair
the DNA damage that is caused by UV light, result in DNA damage
accumulation in XP patients.
They have an increased risk of skin cancers (especially on the face)
Progressive neurological abnormalities
DNA damage cannot be repaired in people suffering from
Cockayne syndrome
Cockayne syndrome is an autosomal recessive condition that can
result from mutations in excision repair proteins (ERCC6 gene or the
ERCC8 gene).
Mutations in DNA repair gene (ATM) cause
ataxiatelangiectasia
Ataxia (loss of motor control) telangiectasia (spider veins) is a rare,
neurodegenerative, autosomal recessive disease and affects the nervous
system, immune system, and other body systems.
The ATM protein which is coded by the ATM gene assists cells in
recognizing damaged or broken DNA strands and coordinates DNA repair
by activating enzymes that fix the broken strands.
DNA double-strand breaks
DNA double-strand breaks can result from:
1) Radiation
2) Reactive chemical
3) Stalled or broken replication forks (most common)
Cells can repair double-strand breaks in one of
two ways
Homologous recombination can flawlessly repair
DNA double-strand breaks
Homologous recombination
Homologous recombination is an exchange of DNA
strands between a pair of homologous duplex DNA
Homologous recombination have many uses in the cell
1) DNA repair
2) Genetic exchange (rearrange DNA sequences)
during meiosis
3) Accurate chromosome segregation during cell
division
Homologous recombination is crucial for meiosis
( ) The replication forks formed at the origin move in opposite directions.
( ) The DNA proofreading activity of a DNA polymerase occurs
concomitantly with strand elongation.
( ) The leading strand telomeres are not completely replicated by DNA
polymerase.
( ) Thymine dimers are covalent links on opposite DNA strands.
( ) Nonhomologous end joining can result in the recovery of
lost nucleotides on a damaged DNA strand.
If the genome of the bacterium E. coli requires about 20 minutes to
replicate itself, how can the genome of the fruit fly Drosophila be
replicated in only 3 minutes?
…… DNA
polymerase
…… single-strand binding protein
…… Okazaki fragment
…… primase
…… sliding clamp
…… RNA primer
…… DNA helicase
Next Class
Next lecture:
RNA and protein synthesis