There are slides about DNA replication and types of DNA.
Here we study about different enzymes of replication and its process.Places of enzyme action also shown in the slides.Different proteins are also discussed.
There are slides about DNA replication and types of DNA.
Here we study about different enzymes of replication and its process.Places of enzyme action also shown in the slides.Different proteins are also discussed.
The process by which DNA molecule makes its identical copies is known as DNA replication or DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule
DNA is maintained in a compressed, supercoiled state.
But basis of replication is the formation of strands based on specific bases pairing with their complementary bases
It is a short and concise slide about DNA replication and Repair. It is prepared keeping in mind for Undergraduates level but PG also might find it handy.
The process by which DNA molecule makes its identical copies is known as DNA replication or DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule
DNA is maintained in a compressed, supercoiled state.
But basis of replication is the formation of strands based on specific bases pairing with their complementary bases
It is a short and concise slide about DNA replication and Repair. It is prepared keeping in mind for Undergraduates level but PG also might find it handy.
The nucleotide structure ,consists of
the nitrogenous base ,attached to the 1’ carbon of deoxyribose
,
the phosphate group attached to the 5’ carbon of deoxyribose
,
a free hydroxyl group (-OH) ,at the 3’ carbon of deoxyribose,1. DNA HELICASES,
to separate the strand,
2. GYRASE (Topoisomerases),
unwind the supercoil,
3. Single strand binding protein (SSBP)
, activity of helicase,
keep two strand separate,
protect DNA from nuclease degradation,
release after replication,
DNA polymerases (DNA manupliation Enzymes).pdfNetHelix
the Secrets of DNA Manipulation: A Comprehensive Exploration of DNA Polymerase and Enzymes
In this PDF presentation entitled "Enzymes that Manipulate DNA, Specially DNA Polymerase," we delve deep into the mechanisms and functions of these remarkable enzymes that play a pivotal role in the realm of molecular biology.
🧬 Key Highlights:
Introduction to DNA Polymerase:
Uncover the fundamental aspects of DNA polymerase, a key player in DNA replication and repair. Explore its structure, functions, and the indispensable role it plays in maintaining the genetic integrity of living organisms.
Types of DNA Polymerases:
Delve into the diverse landscape of DNA polymerases, ranging from prokaryotic to eukaryotic systems. Understand how different types of DNA polymerases contribute to the precision and efficiency of DNA synthesis.
Examples of polymerases:
•DNA polymerase 1
•klenow fragment
•sequenase
•Taq polymerase
•Reverse Transcriptase
DNA Replication
Take a closer look at the intricate dance of enzymes during DNA replication. Follow the step-by-step process, and gain insights into how DNA polymerase ensures the accurate transmission of genetic information from one generation to the next.
Technological Applications:
Unleash the potential of DNA polymerase in various biotechnological applications. From PCR (Polymerase Chain Reaction) to DNA sequencing, discover how these enzymes have revolutionized molecular biology and genetic research.
Emerging Trends and Future Prospects:
Stay ahead of the curve by exploring the latest advancements and emerging trends in DNA manipulation. Witness the ongoing research that promises to unlock new possibilities in the field.
🎓 Who Should Explore This Presentation?
Students and researchers in molecular biology and genetics
Biotechnologists and professionals in the field of genetic engineering
Enthusiasts curious about the molecular machinery behind DNA manipulation
LF Energy Webinar: Electrical Grid Modelling and Simulation Through PowSyBl -...DanBrown980551
Do you want to learn how to model and simulate an electrical network from scratch in under an hour?
Then welcome to this PowSyBl workshop, hosted by Rte, the French Transmission System Operator (TSO)!
During the webinar, you will discover the PowSyBl ecosystem as well as handle and study an electrical network through an interactive Python notebook.
PowSyBl is an open source project hosted by LF Energy, which offers a comprehensive set of features for electrical grid modelling and simulation. Among other advanced features, PowSyBl provides:
- A fully editable and extendable library for grid component modelling;
- Visualization tools to display your network;
- Grid simulation tools, such as power flows, security analyses (with or without remedial actions) and sensitivity analyses;
The framework is mostly written in Java, with a Python binding so that Python developers can access PowSyBl functionalities as well.
What you will learn during the webinar:
- For beginners: discover PowSyBl's functionalities through a quick general presentation and the notebook, without needing any expert coding skills;
- For advanced developers: master the skills to efficiently apply PowSyBl functionalities to your real-world scenarios.
Key Trends Shaping the Future of Infrastructure.pdfCheryl Hung
Keynote at DIGIT West Expo, Glasgow on 29 May 2024.
Cheryl Hung, ochery.com
Sr Director, Infrastructure Ecosystem, Arm.
The key trends across hardware, cloud and open-source; exploring how these areas are likely to mature and develop over the short and long-term, and then considering how organisations can position themselves to adapt and thrive.
Securing your Kubernetes cluster_ a step-by-step guide to success !KatiaHIMEUR1
Today, after several years of existence, an extremely active community and an ultra-dynamic ecosystem, Kubernetes has established itself as the de facto standard in container orchestration. Thanks to a wide range of managed services, it has never been so easy to set up a ready-to-use Kubernetes cluster.
However, this ease of use means that the subject of security in Kubernetes is often left for later, or even neglected. This exposes companies to significant risks.
In this talk, I'll show you step-by-step how to secure your Kubernetes cluster for greater peace of mind and reliability.
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Ramesh Iyer
In today's fast-changing business world, Companies that adapt and embrace new ideas often need help to keep up with the competition. However, fostering a culture of innovation takes much work. It takes vision, leadership and willingness to take risks in the right proportion. Sachin Dev Duggal, co-founder of Builder.ai, has perfected the art of this balance, creating a company culture where creativity and growth are nurtured at each stage.
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
Guy Korland, CEO and Co-founder of FalkorDB, will review two articles on the integration of language models with knowledge graphs.
1. Unifying Large Language Models and Knowledge Graphs: A Roadmap.
https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
https://www.microsoft.com/en-us/research/blog/graphrag-unlocking-llm-discovery-on-narrative-private-data/
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
5. 5
Site-directed mutagenesis
CAG
GTC
CAG
GCC
CAG
GCC
CAG
+ polymerase
+ primer
replication
GCC
CGG
Mutant
Thr
translation
Wild type
GTC
CAG
Val
translation
Only one amino acid changed
Wild type protein
Mutant protein
primer
(1)
(2)
(3)
(5)
(4)
(6)
Val → ThrSmith (1993)
JuangRH(2004)BCbasics
6. 6
AbbreviationsAbbreviations
dsDNA.dsDNA.
ssDNA.ssDNA.
Ori.Ori.
SSBsSSBs
DnaK,dnaJ,dnaEDnaK,dnaJ,dnaE
DNA Helicase.DNA Helicase.
dRibosedRibose
ApoptosisApoptosis
Leading & Laging strands.Leading & Laging strands.
Gyrase.Gyrase.
Speed:100nts/sec. total 9 hours toSpeed:100nts/sec. total 9 hours to
complete in a typical cell.complete in a typical cell.
Double stranded DNADouble stranded DNA
Single stranded DNASingle stranded DNA
Origin of replication.Origin of replication.
Single strand Binding.Single strand Binding.
Heat shock proteins EHeat shock proteins E
Unwind short segmenUnwind short segmen
De-OxyriboseDe-Oxyribose
Programmed cell deathProgrammed cell death
One strech, multiple streches.One strech, multiple streches.
Negative supercoiling using ATPNegative supercoiling using ATP
7. 7
cDNA asingle stranded DNA molecule thatcDNA asingle stranded DNA molecule that
is complementary to mRNA and isis complementary to mRNA and is
synthesised from it by the action ofsynthesised from it by the action of
reverse transcriptase.reverse transcriptase.
miRNAS micro RNAs 21-25 nucleotidemiRNAS micro RNAs 21-25 nucleotide
long.long.
Sines Short interspread repeatSines Short interspread repeat
sequences.sequences.
Si RNA silencing RNA 21-25 nt length canSi RNA silencing RNA 21-25 nt length can
cause gene knockdown.cause gene knockdown.
8. 8
PROTEINS & FUNCTIONSPROTEINS & FUNCTIONS
DNA polymerasesDNA polymerases
Helicases.Helicases.
Topoisomerases.Topoisomerases.
DNA primaseDNA primase
SSB proteinsSSB proteins
DNA LigaseDNA Ligase
Polymerisation.Polymerisation.
Unwinding of DNA.Unwinding of DNA.
Remove supercoiling.Remove supercoiling.
Initiates synth. of RNAInitiates synth. of RNA
primer.primer.
Prevent reanealing ofPrevent reanealing of
dsDNA.dsDNA.
Seals the nick inSeals the nick in
okazaki fragments.okazaki fragments.
9. 9
REQUIREMENTSREQUIREMENTS
Four activated precursors ofFour activated precursors of
dATP,dGTP,dCTP andTTP MgdATP,dGTP,dCTP andTTP Mg++++
..
Template Strand.Template Strand.
Primer with free3’-OH group(10-200)Primer with free3’-OH group(10-200)
Elongation proceeds 5’—3’ direction.Elongation proceeds 5’—3’ direction.
Removal of mismatched nucleotides.Removal of mismatched nucleotides.
Error rate is less than 10Error rate is less than 10 -8-8 per
bp.
3’3’
hydroxyl group attack(nucleophillic) on
po4 of recently attached nucleotide.
10. 10
DNADNA
DNA stands for deoxyribose nucleic acid
This chemical substance is present in the
nucleus of all cells in all living organisms
DNA controls all the chemical changes
which take place in cells
The kind of cell which is formed, (muscle,
blood, nerve etc) is controlled by DNA
The kind of organism which is produced
(buttercup, giraffe, herring, human etc) is
controlled by DNA The kind of organism
which is produced (buttercup, giraffe,
herring, human etc) is controlled by DNA
11. 11
Ribose is a sugar, like glucose, but with only five
carbon atoms in its molecule
Deoxyribose is almost the same but lacks one
oxygen atom
Both molecules may be represented by the
symbol
Ribose & deoxyriboseRibose & deoxyribose
12. 12
The most common organic bases are
Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)
The basesThe bases
13. 13
The deoxyribose, the phosphate and one of the bases
adenine
deoxyribose
PO4
Combine to form a nucleotide
NucleotidesNucleotides
14. 14
A molecule of
DNA is formed by
millions of
nucleotides joined
together in a long
chain
PO4
PO4
PO4
PO4
sugar-phosphate
backbone + bases
Joined nucleotidesJoined nucleotides
17. 17
DNA ReplicationDNA Replication
Priming:Priming:
1.1. RNA primersRNA primers: before new DNA strands can: before new DNA strands can
form, there must be small pre-existingform, there must be small pre-existing
primers (RNA)primers (RNA) present to start the addition ofpresent to start the addition of
new nucleotidesnew nucleotides (DNA Polymerase)(DNA Polymerase)..
2.2. PrimasePrimase: enzyme that polymerizes: enzyme that polymerizes
(synthesizes) the(synthesizes) the RNA PrimerRNA Primer..
18. 18
This DNA polymerase replaces the RNA primer
with DNA.
This is a different type of DNA polymerase
from the main DNA polymerase which
synthesises DNA on a DNA template.
Another DNA polymerase:
19. 19
In E. coli the main enzyme is DNA
polymerase III .
And the enzyme that replaces the RNA
primer with DNA is DNA polymerase I.
When the RNA primer has been
replaced with DNA, there is a gap
between the two Okazaki fragments
and this is sealed by DNA ligase.
20. 20
DNA ligase seals the gap left between Okazaki
fragments after the primer is removed. As the
Okazaki fragments are joined, the new lagging
strand becomes longer and longer.
DNA ligase:
Location: At the replication fork.
Function: Unwinds the DNA double helix.
Helicase:
21. 21
Location: On the template strands.
Function: Synthesizes new DNA in the
5' to 3' direction using the base
information on the template strand to
specify the nucleotide to insert on the new
chain. Also does some proofreading; that
is, it checks that the new nucleotide being
added to the chain carries the correct base
as specified by the template DNA.
DNA polymerase:
22. 22
The new DNA strand made discontinuously
in the direction opposite to the direction in
which the replication fork is moving.
The new DNA strand made continuously in
the same direction as movement of the
replication fork.
Lagging Strand:
Leading strand:
23. 23
If an incorrect base pair is formed,
DNA polymerase can delete the new
nucleotide and try again. In E. coli
the enzyme used for all new DNA
synthesis except for the replacement
of the RNA primers is DNA
polymerase III. DNA polymerase I
replaces the primers.
24. 24
Location: On the template strand which
dictates new DNA synthesis away from the
direction of replication fork movement.
Okazaki fragment:
25. 25
Function: A building block for DNA
synthesis of the lagging strand. On one
template strand, DNA polymerase
synthesizes new DNA in a direction away
from the replication fork movement.
Because of this, the new DNA synthesized
on that template is made in a
discontinuous fashion; each segment is
called an Okazaki fragment.
26. 26
Location: Wherever the synthesis of a new
DNA fragment is to commence.
Function: DNA polymerase cannot start the
synthesis of a new DNA chain, it can only
extend a nucleotide chain primer. Primase
synthesizes a short RNA chain that is used as
the primer for DNA synthesis by DNA
polymerase.
Primase:
27. 27
Location: On single-stranded DNA near the
replication fork. Function: Binds to single-
stranded DNA to make it stable.
Single-strand binding (SSB)
proteins
29. 29
DNA Replication 1
Models of DNA replication: -Meselson-Stahl
Experiment
DNA synthesis and elongation
DNA polymerases
Origin and initiation of DNA replication
Prokaryote/eukaryote models
Telomere replication
30. 30
Let us animate theLet us animate the
process of replicationprocess of replication
33. 33
Sugar phosphate backbone is made by joining the adjcent
nucleotides ( DNA polymarase enzyme( ) )
Nucleotides with Complementary bases are assembled
alongside each strands
35. 35
1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication (Fig. 3.2)
36. 36
1955: Arthur Kornberg
Worked with E. coli.
Discovered the mechanisms of DNA synthesis.
Four components are required:
1. dNTPs: dATP, dTTP, dGTP, dCTP
(deoxyribonucleoside 5’-triphosphates)
(sugar-base + 3 phosphates)
2. DNA template
3. DNA polymerase (Kornberg enzyme)
4. Mg 2+
(optimizes DNA polymerase activity)
1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)
37. 37
Three main features of the DNA synthesis
reaction:
1. DNA polymerase I catalyzes formation of phosphodiester bond
between 3’-OH of the deoxyribose (on the last nucleotide) and
the 5’-phosphate of the dNTP.
• Energy for this reaction is derived from the release of two of the
three phosphates.
2. DNA polymerase “finds” the correct complementary dNTP at each
step in the lengthening process.
• rate ≤ 800 dNTPs/second
• low error rate
3. Direction of synthesis is 5’ to 3’
40. 40
There are many different types of DNA polymerase
Polymerase
Polymerization
(5’-3’)
Exonucleas
e (3’-5’)
Exonuclease
(5’-3’)
#Copies
I YES YES YES 400
II YES NO YES?
III YES YES YES20-40
41. 41
3’ to 5’ exonuclease activity = ability to remove
nucleotides from the 3’ end of the chain
Important proofreading ability
– Without proofreading error rate (mutation rate) is 1
x 10-6
– With proofreading error rate is 1 x 10-9 (1000-fold
decrease)
5’ to 3’ exonuclease activity functions in DNA
replication & repair.
42. 42
Eukaryotic enzymes:
Five DNA polymerases from mammals.
Polymerase α (alpha): nuclear, DNA replication,
no proofreading
Polymerase β (beta): nuclear, DNA repair, no
proofreading
Polymerase γ (gamma): mitochondria, DNA
repl., proofreading
Polymerase δ (delta): nuclear, DNA replication,
proofreading
43. 43
Polymerase ε (epsilon): nuclear, DNA
repair (?), proofreading
Different polymerases for nucleus and
mtDNA
Some polymerases proofread; others do not.
Some polymerases used for replication;
others for repair
44. 44
Origin of replication (e.g., the prokaryote example):
Begins with double-helix denaturing into single-strands thus exposing the
bases.
Exposes a replication bubble from which replication proceeds in both
directions.
45. 45
Initiation of replication, major elements:
Segments of single-stranded DNA are called template
strands.
Gyrase (a type of topoisomerase) relaxes the supercoiled
DNA.
Initiator proteins and DNA helicase binds to the DNA at
the replication fork and untwist the DNA using energy
derived from ATP (adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA
helicase)
DNA primase next binds to helicase producing a
complex called a primosome (primase is required for
synthesis),
46. 46
Primase synthesizes a short RNA primer of 10-12
nucleotides, to which DNA polymerase III adds
nucleotides.
Polymerase III adds nucleotides 5’ to 3’ on both strands
beginning at the RNA primer.
The RNA primer is removed and replaced with DNA by
polymerase I, and the gap is sealed with DNA ligase.
Single-stranded DNA-binding (SSB) proteins (>200)
stabilize the single-stranded template DNA during the
process.
48. 48
DNA replication is continuous on the leading strand and semidiscontinuous on the
lagging strand:
Unwinding of any single DNA replication fork proceeds in one direction.
The two DNA strands are of opposite polarity, and DNA polymerases only
synthesize DNA 5’ to 3’.
Solution: DNA is made in opposite directions on each template.
•Leading strand synthesized 5’ to 3’ in the direction of
the replication fork movement.
continuous
requires a single RNA primer
•Lagging strand synthesized 5’ to 3’ in the opposite
direction.
semidiscontinuous (i.e., not continuous)
requires many RNA primers
49. 49
3
Polymerase III
5’ →
3’
Leading strand
base pairs
5’
5’
3’
3’
Supercoiled DNA relaxed by gyrase & unwound by helicase + pr
Helicase
+
Initiator Proteins
ATP
SSB Proteins
RNA Primer
primase
2Polymerase III
Lagging strand
Okazaki Fragments
1
RNA primer replaced by polymerase I
& gap is sealed by ligase
52. 52
Two Libraries : cDNA Library vs Genomic Library
mRNA
cDNA
Reverse transcription
Chromosomal DNA
Restriction digestion
Genes in expression Total Gene
Complete
gene Gene fragments
Smaller
Library
Larger
Library
Vector:
Plasmid or phage
Vector: Plasmid
Juang RH (2004) BCbasics
53. 53
Restriction Mapping of DNA
A B 10 kb
8 kb
2 kb
A
7 kb
3 kb
B
5 kb
3 kb
2 kb
A
+
B
CK A B A+B M
Restriction
enzymes
Juang RH (2004) BCbasics
54. 54
The Specific Cutting and Ligation of DNA
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
AATTC
G
AATTC
G
G
CTTAA
G
CTTAA
AATTC
G
G
CTTAA
AATTC
G
G
CTTAA
AATTC
G
EcoRI
DNA Ligase
EcoRI sticky end EcoRI sticky end
Juang RH (2004) BCbasics
59. 59
Concepts and terms to understand:
Why are gyrase and helicase required?
The difference between a template and a primer?
The difference between primase and polymerase?
What is a replication fork and how many are there?
Why are single-stranded binding (SSB) proteins required?
How does synthesis differ on leading strand and lagging strand?
Which is continuous and semi-discontinuous?
What are Okazaki fragments?
60. 60
Replication of
circular DNA in
E. coli (3.10):
1. Two replication forks result in
a theta-like (θ) structure.
2. As strands separate, positive
supercoils form elsewhere in
the molecule.
3. Topoisomerases relieve
tensions in the supercoils,
allowing the DNA to continue
to separate.
61. 61
Rolling circle model of DNA
replication (3.11):
1. Common in several
bacteriophages including
λ.
2. Begins with a nick at the
origin of replication.
3. 5’ end of the molecule is
displaced and acts as
primer for DNA synthesis.
4. Can result in a DNA
molecule many multiples
of the genome length (and
make multiple copies
quickly).
5. During viral assembly the
DNA is cut into individual
viral chromosomes.
62. 62
DNA replication in eukaryotes:
Copying each eukaryotic chromosome during the S phase of the cell cycle
presents some challenges:
Major checkpoints in the system
1. Cells must be large enough, and the environment favorable.
2. Cell will not enter the mitotic phase unless all the DNA has replicated.
3. Chromosomes also must be attached to the mitotic spindle for mitosis
to complete.
4. Checkpoints in the system include proteins call cyclins and enzymes
called cyclin-dependent kinases (Cdks).
63. 63
• Each eukaryotic chromosome is one linear DNA
double helix
• Average ~108
base pairs long
• With a replication rate of 2 kb/minute, replicating
one human chromosome would require ~35 days.
• Solution ---> DNA replication initiates at many
different sites simultaneously.
Fig. 3.14
65. 65
(or telomeres What about the ends ) of linear
chromosomes?
DNA polymerase/ligase cannot fill gap at end of chromosome after
RNA primer is removed. this gap is not filled, chromosomes
would become shorter each round of replication!
Solution:
1. Eukaryotes have tandemly repeated sequences at the ends of
their chromosomes.
2. Telomerase (composed of protein and RNA complementary to
the telomere repeat) binds to the terminal telomere repeat and
catalyzes the addition of of new repeats.
3. Compensates by lengthening the chromosome.
4. Absence or mutation of telomerase activity results in
chromosome shortening and limited cell division.
67. 67
Final Step - Assembly into Nucleosomes:
• As DNA unwinds, nucleosomes must disassemble.
• Histones and the associated chromatin proteins must
be duplicated by new protein synthesis.
• Newly replicated DNA is assembled into nucleosomes
almost immediately.
• Histone chaperone proteins control the assembly.
Fig. 3.17
72. 72
1.1. SINGLE BASE ALTERATIONSSINGLE BASE ALTERATIONS
DEPURINATIONDEPURINATION
DEAMINATION OF CYTOSINE TO URACILDEAMINATION OF CYTOSINE TO URACIL
DEAMINATION OF ADENINE TODEAMINATION OF ADENINE TO
HYPOXANTHINEHYPOXANTHINE
ALKYLATION OF BASESALKYLATION OF BASES
INSERTION OR DELETION OF NUCLEOTIDESINSERTION OR DELETION OF NUCLEOTIDES
BASE ANALOG INCORPORATIONBASE ANALOG INCORPORATION
73. 73
2.2. TWO BASE ALTERATIONSTWO BASE ALTERATIONS
UV INDUCED THYMINE-THYMINEUV INDUCED THYMINE-THYMINE
DIMERSDIMERS
BIFUNCTIONAL ALKYLATING AGENTSBIFUNCTIONAL ALKYLATING AGENTS
75. 75
4. CROSS LINKAGE4. CROSS LINKAGE
BETWEEN BASES IN SAME ANDBETWEEN BASES IN SAME AND
OPPOSITE STRANDOPPOSITE STRAND
BETWEEN DNA AND PROTEINBETWEEN DNA AND PROTEIN
MOLECULESMOLECULES
76. 76
1. Proof
reading
and editing
2. Mismatch
Repair
system
3. Base
Excision
repair
4. Nucleotide
Excision
Repair
5. Photo-
Reactivation
Or
Direct
repair
6. Double
Strand
Break
Repair
7.Transcription-
Coupled
repair
77. 77
1. PROOF READING AND1. PROOF READING AND
EDITINGEDITING Despite doubleDespite double
monitoring duringmonitoring during
replication, first at time ofreplication, first at time of
incorporation of bases andincorporation of bases and
second by later follow upsecond by later follow up
energy requiringenergy requiring
processes, some mispairedprocesses, some mispaired
bases persist which havebases persist which have
to be removed by otherto be removed by other
enzyme systems.enzyme systems.
The proof reading activityThe proof reading activity
is carried out by 3’-5’is carried out by 3’-5’
exonuclease activities ofexonuclease activities of
DNA polymerase III andDNA polymerase III and
I.I.
78. 78
2. MISMATCH REPAIR SYSTEM2. MISMATCH REPAIR SYSTEM
This mechanism operates immediately afterThis mechanism operates immediately after
DNA replication.DNA replication.
Sometimes the replication errors escape theSometimes the replication errors escape the
DNA proofreading function. This mechanismDNA proofreading function. This mechanism
checks for the correction of escaped bases.checks for the correction of escaped bases.
Specific proteins scan the newly synthesizedSpecific proteins scan the newly synthesized
DNA by the following mechanisms:DNA by the following mechanisms:
1.1. Identification of mismatched strandIdentification of mismatched strand
2.2. Repair of mispaired base.Repair of mispaired base.
79. 79
In error detection,In error detection,
parent strand isparent strand is
identified first with theidentified first with the
help of GATC-help of GATC-
sequences, that occursequences, that occur
approx. once after everyapprox. once after every
thousand nucleotides.thousand nucleotides.
It is methylated atIt is methylated at
adenine residue.adenine residue.
The methylation doesThe methylation does
not occur immediatelynot occur immediately
after replication. So, theafter replication. So, the
new strand is notnew strand is not
methylated and is easilymethylated and is easily
identified.identified.
80. 80
Secondly, on the new strand, GATC-endonucleaseSecondly, on the new strand, GATC-endonuclease
‘nicks’ the mismatched strand.‘nicks’ the mismatched strand.
This faulty strand is digested by exonuclease.This faulty strand is digested by exonuclease.
An extensive region, from the mismatched area tillAn extensive region, from the mismatched area till
the next GATC-sequence is removed.the next GATC-sequence is removed.
This gap is filled by the DNA polymerase I, in 5’-3’This gap is filled by the DNA polymerase I, in 5’-3’
direction.direction.
Clinical significance-Clinical significance- A defect in mismatch repairA defect in mismatch repair
in humans has been known to cause hereditary non-in humans has been known to cause hereditary non-
polyposis colon cancer (HNPCC).polyposis colon cancer (HNPCC).
81. 81
3. BASE EXCISION REPAIR3. BASE EXCISION REPAIR
This mechanism operates all the time in the cells.This mechanism operates all the time in the cells.
The bases of DNA can be altered:The bases of DNA can be altered:
a)a) SpontaneouslySpontaneously -- cytosine uracil.cytosine uracil.
b)b) Deaminating compounds -Deaminating compounds - like NO, which is formedlike NO, which is formed
from nitrosamines,nitrites, and nitrates.from nitrosamines,nitrites, and nitrates.
- NO is a potent de-aminating compound, that converts:- NO is a potent de-aminating compound, that converts:
i.i. Ctytosine uracilCtytosine uracil
ii.ii. Adenine hypoxanthineAdenine hypoxanthine
iii.iii. Guanine xanthineGuanine xanthine
c)c) Bases can also be lost spontaneously -Bases can also be lost spontaneously - approximately,approximately,
10,000 purine bases are lost spontaneously per cell per10,000 purine bases are lost spontaneously per cell per
day.day.
82. 82
Following mechanismsFollowing mechanisms
operate to correct such baseoperate to correct such base
alterations or losses:alterations or losses:
1.1. Removal of abnormalRemoval of abnormal
bases-bases- Abnormal bases areAbnormal bases are
recognized by specificrecognized by specific
glycosylasesglycosylases..
-they hydrolytically cleave-they hydrolytically cleave
them from deoxy-ribose-them from deoxy-ribose-
phosphate backbone of thephosphate backbone of the
strand.strand.
-this results in either A-this results in either Apurinicpurinic
or Apyrimidinicor Apyrimidinic site,site,
referred to asreferred to as AP- siteAP- site..
83. 83
2.2. Repair of AP-site -Repair of AP-site - AP-endonucleaseAP-endonuclease recognizes therecognizes the
empty site and starts excision by making a cut at 5’-end ofempty site and starts excision by making a cut at 5’-end of
AP-site.AP-site.
-- deoxy-ribose-phosphate lyasedeoxy-ribose-phosphate lyase removes the single, empty,removes the single, empty,
sugar-phosphate residue and gap is finally filled bysugar-phosphate residue and gap is finally filled by DNADNA
polymerase Ipolymerase I and nick is sealed byand nick is sealed by DNA ligaseDNA ligase..
NOTE..NOTE..
By the similar series of steps involving initially theBy the similar series of steps involving initially the
recognition of the defect, the alkylated bases and baserecognition of the defect, the alkylated bases and base
analogs can be removed from DNA. And thus, DNAanalogs can be removed from DNA. And thus, DNA
returns to its original information content.returns to its original information content.
This mechanism is efficient only for replacement of aThis mechanism is efficient only for replacement of a
single base but is not efficient for replacing regions ofsingle base but is not efficient for replacing regions of
damaged DNA.damaged DNA.
84. 84
Recognition and excision ofRecognition and excision of
defectdefect
(eg:-UV induced dimers)(eg:-UV induced dimers)
First,a UV-specificFirst,a UV-specific
endonuclease recognizes theendonuclease recognizes the
dimer and cleaves thedimer and cleaves the
damaged strand atdamaged strand at
phosphodiester bonds on bothphosphodiester bonds on both
5’ side and 3’ side of the5’ side and 3’ side of the
dimer.dimer.
The damaged oligonucleotideThe damaged oligonucleotide
is released, leaving a gap inis released, leaving a gap in
the DNA strand that formerlythe DNA strand that formerly
contained the dimer.contained the dimer.
This gap is filled byThis gap is filled by DNADNA
polymerase Ipolymerase I and nick isand nick is
sealed bysealed by DNA ligaseDNA ligase..
85. 85
5. PHOTO-REACTIVATION OR5. PHOTO-REACTIVATION OR
DIRECT REPAIRDIRECT REPAIR
This is also called ‘This is also called ‘lightlight
induced repair’.induced repair’.
The enzyme photo-The enzyme photo-
reactivating enzyme (PRreactivating enzyme (PR
enzyme) brings about anenzyme) brings about an
enzymatic cleavage ofenzymatic cleavage of
thymine dimers activated bythymine dimers activated by
the visible lightthe visible light
It leads to restoration ofIt leads to restoration of
monomeric condition.monomeric condition.
Co-enzymes required for theCo-enzymes required for the
reaction are FADH2 andreaction are FADH2 and
THF.THF.
86. 86
6. DOUBLE STRAND BREAK6. DOUBLE STRAND BREAK
REPAIRREPAIR High energy radiation, oxidative free radicals or someHigh energy radiation, oxidative free radicals or some
chemotherapeutic agents bring about double-strandedchemotherapeutic agents bring about double-stranded
breaks in DNA, or may also occur naturally duringbreaks in DNA, or may also occur naturally during
naturally during gene rearrangements.naturally during gene rearrangements.
They are potentially lethal to the cell.They are potentially lethal to the cell.
They cannot be repaired by excising single strand andThey cannot be repaired by excising single strand and
using the other strand as template to replace missingusing the other strand as template to replace missing
nucleotides.nucleotides.
It is repaired by 2 ways:It is repaired by 2 ways:
1.1. Non-homologous end-joining repair.Non-homologous end-joining repair.
2.2. Homologous recombination repair.Homologous recombination repair.
87. 87
1.1. Non-homologous end joining repair-Non-homologous end joining repair-
In this system the two ends of DNA areIn this system the two ends of DNA are
brought together by a group of proteins andbrought together by a group of proteins and
thereby the ends are re-ligated.thereby the ends are re-ligated.
This system does not require that the 2 DNAThis system does not require that the 2 DNA
sequences have any homology.sequences have any homology.
2.2. Homologous recombination repair-Homologous recombination repair-
This system uses the enzymes that normallyThis system uses the enzymes that normally
perform genetic recombination betweenperform genetic recombination between
homologous chromosomes during meiosis.homologous chromosomes during meiosis.
This is called sister-strand exchange.This is called sister-strand exchange.
89. 89
7. TRANSCRIPTION COUPLED7. TRANSCRIPTION COUPLED
REPAIRREPAIR
When RNA polymerase transcribes aWhen RNA polymerase transcribes a
gene, as it encounters a damagedgene, as it encounters a damaged
region, the transcription stops.region, the transcription stops.
The excision repair enzymes repairThe excision repair enzymes repair
the area and then transcriptionthe area and then transcription
resumes.resumes.
91. 91
1.1. XERODERMAXERODERMA
PIGMENTOSAPIGMENTOSA
Autosomal recessive in nature.Autosomal recessive in nature.
UV- specific exonuclease is deficient.UV- specific exonuclease is deficient.
Cutaneous hypersensitivity to UV-rays.Cutaneous hypersensitivity to UV-rays.
Blisters on skin.Blisters on skin.
Hyperpigmentation.Hyperpigmentation.
Corneal ulcer.Corneal ulcer.
Death occurs due to formation of cancers ofDeath occurs due to formation of cancers of
skin.skin.
92. 92
2. ATAXIA TELANGIECTASIA2. ATAXIA TELANGIECTASIA
Autosomal recessive inAutosomal recessive in
nature.nature.
Increased sensitivity to X-Increased sensitivity to X-
rays and UV-rays.rays and UV-rays.
Progressive cerebellarProgressive cerebellar
ataxia.ataxia.
Oculo-cutaneousOculo-cutaneous
telangiectasia.telangiectasia.
Frequent sino-pulmonaryFrequent sino-pulmonary
infections.infections.
Lympho-reticular neoplasm.Lympho-reticular neoplasm.
93. 93
3. BLOOM’S SYNDROME3. BLOOM’S SYNDROME
Chromosomal breaks orChromosomal breaks or
rearrangements are seen.rearrangements are seen.
Defect lies in DNA helicase orDefect lies in DNA helicase or
ligase.ligase.
Facial erythmia.Facial erythmia.
Photosensitivity.Photosensitivity.
Lympho-reticularLympho-reticular
malignancies.malignancies.
94. 94
4.FANCONI SYNDROME4.FANCONI SYNDROME
Lethal aplastic anaemia,due to defectiveLethal aplastic anaemia,due to defective
DNA repair.DNA repair.
Cells can not repair interstrand cross-links,Cells can not repair interstrand cross-links,
or damage induced by X-Rays.or damage induced by X-Rays.
96. 96
INHIBITOR OF DNAINHIBITOR OF DNA
REPLICATIONREPLICATION
Anthracyclines cause chainAnthracyclines cause chain
breakageSu.bstances that act directly onbreakageSu.bstances that act directly on
DNA Polymerases eg. Acyclovir inhibitsDNA Polymerases eg. Acyclovir inhibits
the DNA polymerase of herpes simplex.the DNA polymerase of herpes simplex.
2’-dideoxyazidocytidine is a inhibitor of2’-dideoxyazidocytidine is a inhibitor of
bacterial primase,andbacterial primase,and
cournermycin,novobiocin,oxolinic acid andcournermycin,novobiocin,oxolinic acid and
nalidixic acid are effective inhibitor of DNAnalidixic acid are effective inhibitor of DNA
gyrase in bacteria.gyrase in bacteria.
97. 97
TOPOISOMERASE ITOPOISOMERASE I
INHIBITORINHIBITOR
Topoisomerase is essential for DNA replicationTopoisomerase is essential for DNA replication
and cell growth.and cell growth.
Certain drugs produces double strand breaks inCertain drugs produces double strand breaks in
DNA that are irreversible and can lead to cellDNA that are irreversible and can lead to cell
death.death.
Eg. Quilnolne antibiotics,anthracyclines activeEg. Quilnolne antibiotics,anthracyclines active
for treatment of lung, ovarian and colorectalfor treatment of lung, ovarian and colorectal
cancer.The Camptothecins were discoveredcancer.The Camptothecins were discovered
from extract of tree Camptotheca acuminata.from extract of tree Camptotheca acuminata.