A short presentation on DNA damage/mutation and the repair mechanisms involved that I prepared for my online chapter presentation. (The content and pictures credited to Watson et al.'s Molecular Biology of the Gene, 7th Edition)
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
Creative Bioarray provides Cell Apoptosis Assays to all of our customers. The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms.
https://www.creative-bioarray.com/cell-apoptosis-assays.htm
DNA Repair and its cause of emergence. Mutation and its types. Various repair mechanisms in living organisms with its distinctive types along with two common examples: Progeria and Multiple Sclerosis(MS).
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
Creative Bioarray provides Cell Apoptosis Assays to all of our customers. The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms.
https://www.creative-bioarray.com/cell-apoptosis-assays.htm
DNA Repair and its cause of emergence. Mutation and its types. Various repair mechanisms in living organisms with its distinctive types along with two common examples: Progeria and Multiple Sclerosis(MS).
Mutation Repair and DNA Replication.pptxhamzalatif40
In this Presentation Chapter 7 & 8 from the book Advanced Molecular Biology are discussed. Focus has been given to the mutation, its types, mutation repair, Different Repairing mechanisms and DNA Replication is explained with details.
Spontaneous mutations occur naturally without any apparent cause. It arises from a variety of sources- Errors in DNA replication, Spontaneous lesions or by Transposable genetic element. These mutations results in several human diseases.
DNA replication, repair and recombination NotesYi Fan Chen
DNA, replication, repair and recombination Notes based on Molecular biology of the cell. Biology Elite: biologyelite.weebly.com, please use together with the presentation
DNA repair, DNA Mutation, Gene Expression by Dr. Anurag YadavDr Anurag Yadav
Various causes of DNA damage,
Methods of DNA repair for the Damage to the DNA structure,
Gene regulation and Gene Expression in eukaryotes and Prokaryotes.
This presentation will help students to brush up their basic concepts and along with that it will help them to understand what are mutations and what are its causes.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
Toxic effects of heavy metals : Lead and Arsenicsanjana502982
Heavy metals are naturally occuring metallic chemical elements that have relatively high density, and are toxic at even low concentrations. All toxic metals are termed as heavy metals irrespective of their atomic mass and density, eg. arsenic, lead, mercury, cadmium, thallium, chromium, etc.
DERIVATION OF MODIFIED BERNOULLI EQUATION WITH VISCOUS EFFECTS AND TERMINAL V...Wasswaderrick3
In this book, we use conservation of energy techniques on a fluid element to derive the Modified Bernoulli equation of flow with viscous or friction effects. We derive the general equation of flow/ velocity and then from this we derive the Pouiselle flow equation, the transition flow equation and the turbulent flow equation. In the situations where there are no viscous effects , the equation reduces to the Bernoulli equation. From experimental results, we are able to include other terms in the Bernoulli equation. We also look at cases where pressure gradients exist. We use the Modified Bernoulli equation to derive equations of flow rate for pipes of different cross sectional areas connected together. We also extend our techniques of energy conservation to a sphere falling in a viscous medium under the effect of gravity. We demonstrate Stokes equation of terminal velocity and turbulent flow equation. We look at a way of calculating the time taken for a body to fall in a viscous medium. We also look at the general equation of terminal velocity.
Salas, V. (2024) "John of St. Thomas (Poinsot) on the Science of Sacred Theol...Studia Poinsotiana
I Introduction
II Subalternation and Theology
III Theology and Dogmatic Declarations
IV The Mixed Principles of Theology
V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
1. The Mutability
and Repair of
DNA
Sanam Parajuli
Content and Pictures from: Molecular Biology of the
Gene(7th Edition), Watson et al.
2. Introduction
• With every division, a living cell has to copy billions of its base pairs to a
new DNA strand, so, mistakes are unavoidable
• During normal functioning too, a living organism requires proper
expression and functioning of thousands of genes
• Mutations: possible in coding sequences or in flanking sequences
governing gene expression
• Very high rates of mutation in the soma can lead catastrophic incidences of
cancer; too much changes in the germline can lead in progeny not surviving
• But too little changes: no evolution, no speciation, no diversity
• Life and biodiversity: sweet balance between DNA damage and repair
3. Intro…
• Two sources of mutation: Inaccuracy in DNA replication and Chemical Damage to
the genetic material
• Replication errors: because of tautomerization that leads to incorrect pair
readings and although the enzymatic machinery of DNA replication attempts to
correct these errors, some escape detection
• Tautomerization: rearrangement of atoms in a molecule, eg: Adenine and
cytosine from amino to imino form, guanine and thymine from keto to enol form.
Now, A-C pairs and G-T pairs.
• DNA damage: because of finite stability, DNA is subject to damage such as loss of
bases, chemical alteration of bases because of environmental mutagens
• Transposons: Jumping genes (not dealt in this chapter)
4.
5. Consequences of replication errors and DNA
Damage
• 1. Introduction of permanent changes to the DNA (mutations), which can alter
the coding sequence of a gene or its regulatory sequences, effects observed in
the progeny
• 2. Changes to the DNA can prevent it from being used as a template for
replication and transcription, immediate effects observed in the cell itself in
function and survival
• Challenge for a cell: scan the genome for errors and also mend the errors if
possible
• How is the DNA mended rapidly enough to prevent setting of mutations? How
are parent and daughter strands distinguished in repair? How does the cell
restore the proper DNA sequence when, because of a break or severe lesion, the
original sequence can no longer be read? How does the cell cope with lesions
that block replication?
• It depends on the type of error to be repaired.
6. Replication Errors and their Repair
The Nature of Mutations
• Simplest kinds: switches of one base for another:
Transitions and Transversions
• Point mutations: alter a single nucleotide
• Other kinds like extensive insertions and deletions can
cause more drastic changes to the DNA
• Ex: insertion of a transposon; rearrangement of
chromosomes; aberrant recombination events
• Probability that new mutations arise spontaneously at any
given site on the chromosome: 10-6 to 10-11 per round of
DNA replication
• Some chromosome sites can be “hotspots” for mutations
while some can mutate less frequently
7. *DNA Microsatellites*
• repeats of simple di-, tri-, or tetranucleotide sequences
• Really prone to mutations
• Ex: CA stretches found scattered across chromosomes
• DNA replication at such sites prone to “slippage” and a population, thus,
can have polymorphic CA stretches at a given point
• Such polymorphism used as physical marker for mapping inherited
mutations
• Triple repeat expansions associated diseases: Fragile X (CGG repeats more
than 200), Huntington’s (CAG repeats coding for excess glutamine) etc
8. Some replication errors escape proofreading
• The 3’5’ exonuclease in the DNA polymerase: removes wrongly incorporated
nucleotides with high accuracy. This proofreading increases the fidelity of DNA
replication by a factor of nearly 100
• However, not foolproof
• Some misincorporated nucleotides escape detection and become a mismatch
between the template and new strand
• For each nucleotide, there are 3 possible mismatches (eg: for T, T:C, T:G, and T:T
are mismatches). So, there are 12 possible mismatches for the 4 nucleotides
• If misincorporated nucleotides not detected and corrected, the sequence change
will be permanent in the genome
9.
10. Mismatch Repair System Removes Errors that
Escape Proofreading
• Fortunately, a mismatch repair system exists for correcting errors that
escape exonuclease repairing that increases the replication accuracy
by an additional 2 or 3 orders of magnitude
• Challenge for this system: First: scanning for mismatches. Mismatches
are transient, only true for the cell of origin, in the daughter cell, it
becomes permanent
• Second: only the daughter strand needs repair, not the parent. How
does the system know which strand to repair?
11. Mismatch Repair System in E. coli
• In E. coli, mismatches are repaired by the dimer of the mismatch repair protein, MutS
• Mismatches cause distortions in the DNA strand, MutS scans for those
• MutS embraces the mismatch containing DNA, induces a pronounced kink in the DNA
and a conformational change in itself
• MutS has an ATPase activity, but its role in repair is not properly understood
• The MutS-mismatched DNA complex recruits MutL, another protein of the repair
complex
• MutL activates MutH, an enzyme that induces a nick in one strand near the site of the
mismatch
• Nicking is followed by unwinding of the DNA towards the site of mismatch by a specific
DNA helicase UvrD and an exonuclease progressively digests the single displaced strand
• The gap is filled by DNA Pol III and sealed with DNA ligase
12.
13.
14. How does the system know which strand to
repair?
• a wrong decision can lead to permanent setting of the mutation
• The solution in E. coli: Transient Hemimethylation
• Dam methylase methylates A residues on both strands of the sequences 5’-GATC-
3’, which is widely distributed in the E. coli genome, about once every 256bp.
• All these sites methylated
• When the replication fork passes a section with both strands methylated, the
resulting daughter duplexes will be hemimethylated, ie methylated only on the
parental strand.
• Hence, until Dam methylase catches up and methylates the daughter strand, the
new strand is marked by the absence of a methyl group, and hence can be
recognized for repair
• MutH can bind at such hemimethylated sites
15.
16. • MutH binds at unmethylated strand and initially its exonuclease
activity is normally latent
• Only when MutH is contacted by MutL and MutS located at a nearby
mismatch (which is likely to be within a distance of a few hundred
base pairs) does MutH become activated
• How they interact in such long distances is uncertain, but evidence
suggests that the MutL-MutS complex leaves the site of mismatch
and moves along the strand to reach MutH at the site of
hemimethylation
• MutH then nicks the unmethylated strand selectively
17. • Still, removal of single stranded DNA between the nick site and the
mismatch site left to be done
• For this, different exonucleases are utilized depending on the site of
the mismatch with respect to the nick
• If the DNA is cleaved on the 5’ side of the mismatch, exonuclease VII
or RecJ, which degrades DNA from 5’3’ direction is used
• If the nick is on the 3’ side, exonuclease I does the job
• DNA Pol III fills in the missing sequence
18.
19. In Eukaryotes?
• Similar mechanism using homologs of MutS (called MSH, or MutS Homologs) and
MutL (called MLH and PMS)
• Eukaryotes have multiple MutS like proteins with different specificities
• Ex: one specific for simple mismatches while other recognized small indels
resulting from “slippage”
• MutH is absent in eukaryotes as well as hemimethylation and most other bacteria
are unable to utilize hemimethylation and lack Dam methylase as well
• Lagging strands produce Okazaki fragments, and before ligation, the space
between these fragments serve as nicks
• Human homologs of the of the MutS (MSH) interact with the sliding-clamp
component of the replisome and would thereby be recruited to the site of
discontinuous DNA synthesis on the lagging strand.
• Interaction with the sliding clamp could also recruit mismatch repair proteins to
the 3’ (growing) end of the leading strand
20. DNA Damage
Spontaneous Damage by Hydrolysis and Deamination
• Chemical/physical mutagens can cause DNA damage
• But DNA cam also go spontaneous damage from action of water (although aq.
Environment is needed for proper double helix)
• Most frequent and important kind: Deamination of the base cytosine
• Under normal physiological conditions, cytosine can go spontaneous deamination
and thus generating Uracil, which is unnatural in DNA
• C would have paired with a G, but now U pairs with A
• Similarly, deamination converts adenine to hypoxanthine, which bonds with
cytosine, and guanine is converted to xanthine, which pairs with cytosine, albeit
with only 2 H-bonds
• DNA also undergoes depurination by spontaneous hydrolysis of the N-glycosyl
linkage, and this produces an abasic site
21. • All these changes create unnatural
changes in the DNA
• The presence of unnatural base makes it
possible for the repair system to recognize
errors
• Evolutionary significance of Thymine
being present in place of Uracil in DNA: If
Uracil was a natural base, deamination of
cytosine would form another natural base,
impossible for the repair mechanism to
detect and correct
22. What if deamination gave rise to natural
bases?
• If deamination gives rise to natural bases, the repair system would not be
able to detect them
• For example: 5-methylcytosine is really common in vertebrate DNA.
• Its deamination produces Thymine (a natural base), not Uracil
• So, the change is not detected by the repair system
• The Thymine will pair then pair with Adenine while forming a new
daughter strand, leading to permanent fixation of the mutation (called C to
T Transition).
• In fact, methylated Cs are hotspots for spontaneous mutations in
vertebrate DNA
23. DNA is damaged by alkylation, oxidation, and
radiation
Alkylation
• Transfer of methyl or ethyl groups to
reactive sites on the bases and to
phosphates on the DNA backbone
• Alkylating chemicals: nitrosamines
and N-methyl-N1-nitro-N-
nitrosoguanidine, a potent lab
mutagen
• Most vulnerable site for alkylation:
keto group at the C-6 of guanine,
which forms O6-methylguanine,
which mispairs with Thymine,
changing G:C pairs to A:T pairs in
daughter cells
24. Oxidation
• DNA is subject to damage from reactive oxygen species as well (O2
-, H2O2, •OH)
• Generated by ionizing radiation and by chemical agents that generate free
radicals
• Oxidation of G forms 7,8-dihydro-8-oxoguanine, or oxoG, which is highly
mutagenic as it can base pair with A as well as C. If it pairs with A during
replication, it gives rise to a G:C to T:A transversion, one of the most common
mutations in human cancers
• Maybe this is one primary way ionizing radiation and oxidizing agents cause
cancer
25. Radiation
• Radiation of nearly 260nm (UV) strongly absorbed
by the bases, one consequence of which can be
photochemical fusion of two adjacent pyrimidines
on the same chain
• If two thymines fuse, a thymine dimer is formed,
which contains a cyclobutane ring generated by
links between carbon atoms 5 and 6 of adjacent
thymine molecules
• If C is adjacent to T, a T-C adduct is formed linking
C6 of T to C4 of C
• Such dimers are incapable of pairing and stop the
movement of DNA polymerase during replication
• Assays to detect DNA damage: Immunoblotting,
Comet Assay (Single cell electrophoresis), Cell
survival assay.
26. Radiation: X and γ-rays
• Ionizing radiation can introduce double strand breaks in the DNA and
severely damage the chromosome
• Can directly attack the backbone of the DNA or create ROS which, in
turn, react with the deoxyribose subunits
• Cells require intact chromosomes to replicate their DNA, so targeted
ionizing radiation is used to damage the DNA of cancer cells
• Anticancer drugs like Bleomycin cause breaks in DNA. Such agents are
called to be clastogenic (Greek clastos, which means broken)
27. Mutations Caused by Base Analogs and
Intercalating Agents
• Base analogs: structurally similar to
proper bases but differ in ways that make
them treacherous to cell, thus can get
taken up by cells, converted to NTPs, and
incorporated into the DNA during
replication, but base-pairing occurs
inaccurately, leading to frequent mistakes
• 5-bromouracil: one of the most mutagenic
analog of Thymine
• It mispairs with Guanine via the enol
tautomer (In case of Thymine, the keto
tautomer is more favored, but for 5-
bromouracil, the enol tautomer is more
favored)
28. • Intercalating agents: Flat molecules with
several polycyclic rings that bind to the
equally flat purine or pyrimidine bases
• They may cause insertions or deletions in
strands
• Insertion: The DNA polymerase reads the
inserted area as more than one base pair,
hence, inserts more bases opposite to it
than actually needed
• Deletion: DNA polymerase skips the
intercalated region and in the process, the
base that the agent had bound to
29. Repair and Tolerance of DNA Damage
• DNA damage can have two types of consequences:
1. Thymine dimers (irradiation), nicks, breaks in the DNA backbone
impede replication or transcription
2. Other kinds of damage (base analogs, intercalation, alkylation,
oxidation, deamination) can cause permanent alterations in the
DNA sequence of the progeny cells
• Cells have evolved elaborate mechanisms to identify and repair DNA
damage before it blocks replication or causes a mutation
30. Direct Reversal of DNA Damage
1. Photoreactivation: DNA photolyase catches energy from light and
uses it to beak down the covalent bonds linking adjacent
pyrimidines (dimers)
31. Direct Reversal of DNA Damage
2. Removal of methyl group from methylated base O6-methylguanine
that pairs with T (resulting from alkylation) by the enzyme
methyltransferase which transfers the methyl group to one of its
cysteine residues (after that, the enzyme is not used again)
32. Base-Excision Repair and Base Flipping
Mechanism
• The most common way of “cleansing” DNA is to remove and replace the
altered base
• Two principal ways: Base Excision Repair and Nucleotide Excision Repair
• In Base Excision Repair, an enzyme called glycosylase recognizes and
removes the damaged base by hydrolysing the glycosidic bond.
• The resulting abasic sugar is removed from the DNA backbone in a further
endonucleolytic step. Endonucleolytic cleavage also removes apurinic and
apyrimidinic sugars that arise by spontaneous hydrolysis
• a repair DNA polymerase and DNA ligase restore an intact strand using the
undamaged strand as a template
33.
34. There are more than 1 type of DNA
Glycosylase
• Depending upon the damaged base to be repaired, there are different
kinds of DNA Glycosylases, 11 have been identified in human cells
• A specific glycosylase recognizes uracil (generated because of
deamination of cytosine), and another is responsible for removing
oxoG (generated because of oxidation of guanine)
35. How do DNA Glycosylases Detect Damage?
• Each base is buried deep in the DNA helix
• Evidence shows that the enzyme diffuses laterally along the minor groove of the
DNA until a specific kind of lesion is detected
• DNA molecule shows great flexibility, the damaged base is flipped out away from
the helix where it sits in the specificity pocket of the glycosylase as seen in X-ray
crystallography
• Base flipping is done without much structural changes to the DNA structure, and
hence, the energetic costs may not be all that much
• Nevertheless, it is unlikely that glycosylases flip out every base to check for
abnormalities as they diffuse along DNA. Thus, the mechanism by which these
enzymes scan for damages is still unknown
36.
37. What if the damaged base is not removed
before replication?
• Fail-safe glycosylase
• Ex: in oxoG:A pairing, if the oxoG base is not removed before
replication, a fail-safe glycosylase removes the A that it pairs with in
the daughter strand and replaces it with C
• Similarly, 5-methylcytosine gives Thymine upon deamination, which
can incorrectly pair with G
• The glycosylase system assumes, so to speak, that the T in a T:G
mismatch arose from deamination of 5-methylcytosine and
selectively removes the T so that it can be replaced with a C.
38.
39. Nucleoside Excision Repair
• This system works by recognizing distortions to the shape of the
double helix, such as those caused by a thymine dimer or by the
presence of a bulky chemical adduct on a base, rather than by
recognizing a specific base
• A chain of events removes a short ssDNA patch that includes the
lesion, and the gap is filled by DNA polymerase
• Accomplished by 4 protein UvrA, UvrB, UvrC, and UvrD, types of
exonucleases
40. • A complex of two UvrA and UvrB molecules scans the DNA, the UvrA subunits
detect the distortions
• After detection, UvrA subunits leave the complex, the remaining UvrB complex
melts the DNA and creates a ss bubble around the lesion
• The UvrB dimer recruits UvrC, UvrC creates two incisions, one located 4 or 5
nucleotes 3’ of the lesion, and the other 8 nucleotides 5’ to the lesion
• The 12-13 residue long lesion containing DNA is removed from the rest of the
DNA by a helicase UvrD
• Uvr: because this system repairs damage from UV light, mutants of the uvr gene
are sensitive to UV light and lack the ability to remove T-T dimer or T-C adducts,
and bulky lesions of many kinds
• In humans, xeroderma pigmentosum genetic disease results from a mutation in
uvr genes or inability to repair UV induced damage, which renders them very
sensitive to sunlight, making them prone to skins lesions, and even cancer
41.
42. • In higher cells, the principle behind nucleotide excision repair is largely the same
as in E. coli
• But the machinery involved is a little more complicated
• Repair and excision involves 25 or more polypeptides (enzymes)
• XPC: equivalent to UvrA (detecting distortions in the DNA)
• XPA and XPD: equivalent to UvrB (forming bubble around the lesion) with the ssb
protein RPA
• The bubble creates a cleavage site for a nuclease called ERCC-1 XPF
5’ to the lesion and 3’ for the nuclease XPG (eq. to UvrC)
• The excised strand is 24-32 nucleotides long, and as in bacteria, gap filling is done
by polymerase and ligase
43. Transcription Coupled Repair
• Nucleotide Excision Repair also rescues RNA polymerase, the progress
of which has been halted due to a distortion in the template strand
• NER proteins recruit to the stalled RNA polymerase during
transcription
• In this effect, RNA polymerase acts as a damage sensing protein
• The TFIIH protein that unwinds the DNA during transcription, includes
two subunits, which are in fact XPA and XPD
• So, helicases of TFIIH melt the DNA around a lesion during NER and
also during transcription
44.
45. Double-strand break (DSB) repair pathways
• How do cells repair double-strand breaks in DNA in which both strands of the
duplex are broken?
• DSB repair pathways accomplish this
• One recombination based pathway retrieves sequence information from the
sister chromosome. Recombination based DSB pathway (another chapter in the
book)
• DNA recombination also helps repair errors in DNA replication (by retrieving seq
information from another daughter molecule of the replication fork and
completing the recombination after which the NER does its job
• or by producing a break while passing over a nick, which can be then repaired by
DSB repair pathways
• Maybe recombination evolved to repair DNA damage as its primary function
46. DSBs in DNA Are Also Repaired by Direct
Joining of Broken Ends
• A DSB is the most cytotoxic of all kinds of DNA damage
• Consequences if left unchecked: blocking replication, chromosome loss, ultimately death
of the cell or neoplastic transformation
• DNA damage repair pathways are overlapping, so recombination alone is not responsible
for mending DSBs
• Recombination relies on sister chromatid’s template for repairing, but what if a non-
replicated chromosome suffers a break?
• An alternative DSB repair system comes into play: Non-Homologous End Joining
• NHEJ is a backup system in yeast, which primarily relies on recombination bases DSB
repair, but in higher cells, it is the primary way
• NHEJ protects and processes the broken ends and joins them together, but broken ends
lose sequence info. Thus NHEJ is mutagenic, but the effects are far less hazardous to the
cell than there are consequences to leaving broken ends unrepaired
47. How NHEJ works
• It doesn’t involve extensive stretches of homologous sequences
• Two ends of the broken DNA are joined by misalignment between single strands
protruding from the broken ends
• Misalignment because of pairing between tiny stretches of complementary bases,
nucleases remove the tails and polymerase fills the gaps
• Till date, seven proteins of the NHEJ pathway have been identified in mammalian
cells: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, Cernunnos-XLF, and DNA ligase IV
• Ku70 and Ku80 are the most fundamental components: constitute a heterodimer
that binds to the DNA ends and recruits DNA-PKc (protein kinase)
• DNA-PKcs, in turn, forms a complex with Artemis (a 5’-3’ exonuclease that is
activated by phosphorylation by DNA-PKcs
• These nucleolytic activities process the broken ends and prepare them for
ligation.
• Ligase IV performs ligation in a complex with XRCC4 and Cernunnos-XLF
48.
49. NHEJ in bacteria
• NHEJ less frequently observed in bacteria
• In Bacillus subtilis, a Ku-like protein and a DNA ligase is produced when it sporulates and it
packages the protein into a mature spore
• This two protein NHEJ system repairs heat-induced DNA breaks when the spore germinates
• Mutants lacking these spores barely survive harsh conditions
• Spores have only one chromosome, so it makes sense that DNA breaks are mended by
NHEJ, not recombination based methods
• The doughnut like structure of the spore chromosome makes the broken ends lie close to
each other, facilitating joining
• NHEJ is the reason why B. subtilis spores are able to survive harsh environments
50. Tolerance of DNA Damage: Translesion
Synthesis
• DNA repair systems are not foolproof and damaged bases can be
encountered by a DNA Pol during replication
• If replication ceased, it can be more damaging
• A fail-safe mechanism to bypass these damages and “tolerate” them
• One mechanism: Translesion Synthesis
• A highly error prone mechanism but spares the cell a much worse fate
of incompletely replicated chromosome
• Tolerance leaves the lesion intact, which can be corrected later by
other pathways
51.
52. Translesion Synthesis
• Catalyzed by a special class of DNA polymerases that synthesize DNA
directly across the site of damage
• In E. coli, DNA Pol IV (DinB) or DNA Pol V (complex of proteins UmuC
and UmuD’) perform the job
• DinB and UmuC are part of a distinct family of DNA polymerases
called Y family
• In humans, out of 5 known translesion polymerases, 4 belong to the Y
family
53.
54. • Translesion polymerases incorporate nucleotides independent of base
pairing, that is why synthesis can occur over a damaged strand
• As they do not read from the template, the process is highly error-prone
• Even for apurinic or apyrimidinic sites in the template, nucleotides are
incorporated
• However, not completely random
• DNA Pol η correctly inserts two A residues opposite a thymine dimer
• Structural studies show that the active site of DNA Pol η is more
accommodating of thymine dimers than DNA Pol κ, another translesion
polymerase
55.
56.
57. • Because of high error rate, transleion synthesis and NHEJ are systems of last
resort
• The price paid for allowing a cell to survive and reproduce: high levels of
mutagenesis (fixed in the genome)
• So, synthesis of translesion polymerases an enzymes are highly regulated
• Ex: in E. coli, these polymerases are synthesized only in response to DNA damage
• SOS Response: DNA Damage leads to the proteolytic destruction of a
transcriptional repressor (the LexA repressor) that controls expression of genes
involved in the SOS response, including those for DinB, UmuC, and UmuD
(precursor of UmuD’). The same pathway is also responsible for the proteolytic
conversion of UmuD to UmuD’
• Cleavage of both LexA and UmuD is stimulated by a protein called RecA, which is
activated by single-stranded DNA resulting from DNA damage. RecA is a dual-
function protein that is also involved in DNA recombination
58. How a translesion polymerase gains access to the
stalled replication machinery at the site of DNA damage
• In mammalian cells, entry into the translesion synthesis pathway is triggered by
chemical modification of the sliding clamp
• Ubiquitination of the sliding clamp: this makes the sliding clamp recruit a translesion
polymerase (it contains domains that recognize and bind to ubiquitin)
• The translesion polymerase then displaces the replicative polymerase from the 3’ end
of the growing strand and extends it across the site of the damage
• Ubiquitination is a distress signal which helps rescue a stalled replication machine
• In addition to this polymerase switching mechanism, a synthesis that used gap filling
mechanism has also been reported
• The replicative DNA polymerase skips over the damage introducing a gap, which is
filled by the translesion polymerase
59.
60. Unexplained questions:
• How exactly does the translesion
enzyme replace the normal replicative
polymerase in the DNA replication
complex?
• How does the normal replicative
polymerase switch back to and replace
the translesion enzyme at the replication
fork? (maybe the translesion enzyme has
low processivity)
Tautomerization: rearrangement of atoms in a molecule, eg: Adenine and cytosine from amino to imino form, guanine and thymine from keto to enol form. Now, A-C pairs and G-T pairs.
DNA lesion refers to a section of a DNA molecule containing a primary damaged site i.e. a base alteration, a base deletion, a sugar alteration or a strand break