DNA is the genetic material that defines every cell. Before a cell duplicates and is divided into new daughter cells through either mitosis or meiosis, biomolecules and organelles must be copied to be distributed among the cells. DNA, found within the nucleus, must be replicated in order to ensure that each new cell receives the correct number of chromosomes. The process of DNA duplication is called DNA replication. Replication follows several steps that involve multiple proteins called replication enzymes and RNA. In eukaryotic cells, such as animal cells and plant cells, DNA replication occurs in the S phase of interphase during the cell cycle. The process of DNA replication is vital for cell growth, repair, and reproduction in organisms.
DNA replication is an important process which takes place in every organisms, be it prokaryotic or eukaryotic. The DNA replication process produces two identical copies of daughter DNA molecules using the existing DNA molecule as template. Each daughter DNA molecule inherits one strand from the parent cell and the other strand is newly synthesized. This is known as semiconservative mode of replication, demonstrated by Meselson and Stahl.
DNA replication is an important process which takes place in every organisms, be it prokaryotic or eukaryotic. The DNA replication process produces two identical copies of daughter DNA molecules using the existing DNA molecule as template. Each daughter DNA molecule inherits one strand from the parent cell and the other strand is newly synthesized. This is known as semiconservative mode of replication, demonstrated by Meselson and Stahl.
DNA replication is the process by which DNA makes a copy of itself during cell division.The separation of the two single strands of DNA creates a 'Y' shape called a replication 'fork'. The two separated strands will act as templates for making the new strands of DNA.
this is an informative presentation regarding the replication of genetic material in prokaryotic cell. it might be useful for individual who is interested in genetics or molecular biology.
Replication:
DNA replication is the biological process of producing two identical copies of DNA from the original/parentral DNA molecule.
This process occurs in all living organism.
Basis for biological inheritance
DNA Replication Is Semiconservative
Replication Begins at an Origin and Usually Proceeds Bidirectionally
DNA Synthesis Proceeds in a 5’-3’ Direction and Is semidiscontinuous
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
“This structure has novel features which are of considerable biological interest.”
This may be the science most famous statement, which appeared in April 1953 in the scientific paper where James Watson and Francis Crick presented the structure of the DNA-helix.
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
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.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
DNA replication is the process by which DNA makes a copy of itself during cell division.The separation of the two single strands of DNA creates a 'Y' shape called a replication 'fork'. The two separated strands will act as templates for making the new strands of DNA.
this is an informative presentation regarding the replication of genetic material in prokaryotic cell. it might be useful for individual who is interested in genetics or molecular biology.
Replication:
DNA replication is the biological process of producing two identical copies of DNA from the original/parentral DNA molecule.
This process occurs in all living organism.
Basis for biological inheritance
DNA Replication Is Semiconservative
Replication Begins at an Origin and Usually Proceeds Bidirectionally
DNA Synthesis Proceeds in a 5’-3’ Direction and Is semidiscontinuous
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
“This structure has novel features which are of considerable biological interest.”
This may be the science most famous statement, which appeared in April 1953 in the scientific paper where James Watson and Francis Crick presented the structure of the DNA-helix.
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
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.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
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.
2. – Anabolic polymerization process that requires
monomers and energy
• Triphosphate deoxyribonucleotides serve both functions
– Key to replication is complementary structure of the
two strands
– Replication is semiconservative
• New DNA composed of one original and one daughter
strand
4. DNA Replication in Prokaryotes
– Initial processes in replication
• Bacterial DNA replication begins at the origin
• DNA polymerase replicates DNA only 5′ to 3′
• Because strands are antiparallel, new strands are synthesized
differently
– Leading strand synthesized continuously
– Lagging strand synthesized discontinuously
5. • Replication begins at a site called Origin (A-T rich region)
• Origin of E.coli is called oriC
• A no. of proteins bind at origin to initiate replication.
• Once initiated, replication proceeds outward from the
origin in both directions- bidirectional
• The where a pair of replicated segments come together and
join the non-replicated DNA-replication fork
7. DNA polymerase
• Major enzymes in replication
• All DNA polymerases polymerize a polynucleotide by adding to an
existing double-stranded stretch of DNA.
• In E. coli- three DNA polymerases: DNA pol I, II & III.
• DNA poly I- found in abundance - involved in DNA repair and
assists with primary DNA replication.
• DNA poly II is exclusively involved in repair.
• DNA poly III is the major DNA polymerase role.
• The degree to which the enzyme remains associated with the
template through successive cycles of nucleotide addition is
referred to as its processivity
8. Different types of DNA polymerases in Prokaryotes
Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’) Funtion
I PolA Yes Yes Yes Repair
II PolB Yes Yes No Repair
III PolC Yes Yes No Replicase
IV dinB repair
V
•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.
9. Other Proteins
• DnaA
• An origin-binding protein.
• It binds cooperatively to the four 9-bp repeats in oriC.
• The origin DNA wrapped around an assembly of 10-20 monomers of
DnaA complexed with ATP.
• An open complex forms when the three AT-rich 13-bp repeats in oriC
unwind as a consequence of the DNA wrapping around the
assembly of DnaA.
• DnaA then guides the DnaB (helicase) hexameric protein from a
DnaB-DnaC complex in solution to its places around each strand.
10. • DnaB (helicase)
• unwinds DNA strands using ATP energy and moves
processively in the 5'-to-3' direction along DNA.
• DnaA together with the use of ATP energy is required to load
DnaB (helicase) onto DNA in the form of a DnaB-DnaC complex.
• After loading DnaB onto the replication fork, DnaC is released
from the DnaB-DnaC complex and leaves the DNA.
11. • DnaC
• forms a complex with DnaB.
• It is required for loading DnaB onto DNA.
• DnaG (Primase)
• Makes RNA primers (about 10 nucleotides long) that are used by
DNA pol III holoenzyme to start DNA synthesis.
• DnaG acts distributively (does not remain associated with DNA).
• It drops off DNA after primer synthesis, then reloads onto DNA a
second or so later by protein-protein interactions with DnaB to
synthesize the next primer on the lagging strand.
12. • SSB (single-strand binding protein)
• does not itself unwind DNA, but binds to and stabilizes
unwound single-stranded DNA
• Gyrase (Topoisomerase II)
• The overwinding of double stranded DNA is relieved by
gyrase.
• Gyrase uses ATP energy to introduce negative
supercoiling into the DNA.
• Gyrase can be considered as the SWIVEL for
replicating molecules.
13. • DNA pol I
• Required to remove RNA primers by simultaneous
action of 5'-to3' exonuclease and DNA polymerase
(nick translation).
• DNA Ligase
• Required to join Okazaki fragments together, uses
NAD+ as energy cofactor.
14. • The unwinding reaction is driven by helicases, a class of proteins
that catalyze the ATP-dependent unwinding of DNA double
helices.
• Helicase requires a single-stranded region for binding.
• It then moves along the DNA strand, its translocation coupled to
ATP hydrolysis and to strand unwinding.
• SSB (ssDNA-binding protein) binds to the unwound strands,
preventing their re-annealing.
• Unlike topoisomerases that alter the linking number of dsDNA
through phosphodiester bond breakage and reunion, helicases
simply disrupt the hydrogen bonds that hold the two strands of
duplex DNA together.
15.
16. 1. Many copies of dnaA bind the four 9-mers; DNA wraps around
dnaA forming “Initial Complex”. This requires ATP and a protein
Hu that is already bound to the DNA.
3. Two copies of dnaB (helicase) bind the 13-mers. This requires
dnaC (which does not remain with the Prepriming Complex) and
ATP.
4. Primase binds to dnaB (helicase) and the DNA.
2. This triggers opening of the 13-mers (Open complex).
5. dnaB: primase complex moves along the template β’>5’
synthesizing RεA primers 5’>β’ for Pol III to extend.
Order of events at OriC
17. Machinery operating at replication fork
• Helicase and SSB proteins unwind DNA
• DnaB helicase is a ring shaped protein (6 sub units)
encircles a single DNA strand.
• DnaB is loaded onto the origin with the help of DnaC
and translocates in 5’-β’ direction along the lagging
strand template, unwinding the helix.
• Primase synthesize RNA primers
• In E.coli primase and helicase associate transiently to
form primosome.
18. • One of the non-catalytic components of DNA pol III holoenzyme (
clamp) keeps the pol associated with DNA template and slide
freely along it.
• Assembly of clamp around DNA requires a multisubunit clamp
loader -a part of pol III.
• In ATP-bound state, the clamp loader binds to primer-template
junction, while loading clamp.
• Once DNA is squeezed through the opening in the clamp wall, ATP
is hydrolyzed, causing the release of clamp, which closes around
the DNA
22. • Evidences suggest that the same DNA pol III molecule
synthesizes the successive fragments of lagging strand.
• For this Pol III is recycled from the site where it just complete
okazaki fragment to the next site.
• The enzyme does this by “hitching a ride” with the DεA pol that
is moving in the leading strand template.
• Even though they move in opposite direction, they are the part
of a single protein complex.
23.
24.
25.
26. Termination
• Diametrically opposite from oriC on the E. coli circular map is
a terminus region, the Ter, or t, locus- act as terminators
• The bidirectionally moving replication forks meet here and
replication is terminated.
• The Ter region contains a number of short DNA sequences
containing a consensus core element 5'-GTGTGTTGT.
27. • Clusters of three or four Ter sequences are organized into
two sets inversely oriented with respect to one another.
• One set blocks the clockwise-moving replication fork, and its
inverted counterpart blocks the counterclockwise-moving
replication fork.
28. • Ter sequence will impede replication only if oriented in the
proper direction with respect to the approaching replication fork
• Also if a specific 36-kD replication termination protein, Tus
protein, is bound to it.
• Tus protein is a contrahelicase.
• Tus protein prevents the DNA duplex from unwinding by
blocking progression of the replication fork and inhibiting the
ATP-dependent DnaB helicase activity.
29. • Replication usually leaves the circular progeny
chromosomes intertwined by 20 to 30 coils about each
other, a so-called catenated state.
• In order to disengage the individual duplexes from each
other prior to their distribution to daughter cells, double-
stranded cuts must be made so that the double helices can
pass through one another.
• Topoisomerase II (DNA gyrase) can catalyze this process.
30.
31. Termination of DNA replication
• The terminus (ter) of DNA replication is opposite the origin of
replication on the circular E. coli chromosome, spanning 450 kb
• Ter is a "trap”: replication forks enter, but do εOT leave this region.
There are six ter sites in this region.
• A protein called Tus binds to the ter sites, and this binding stops
DnaB (helicase).
• Leading strand synthesis terminates one nucleotide away from bound
Tus.
35. Replication of circular DNA in
E. coli (3.10):
1. Two replication forks result in a
theta-like () 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.
36. Fidelity of DNA replication
• In E.coli, chances of incorporating a wrong nucleotide during
replication is <10-9
• If the incoming nucleotide is correct, a conformational change
occurs in which the fingers of the pol rotate towards the palm
gripping the incoming nucleotide.
• If the newly formed pair exhibits improper geometry, the active
site of Pol can not achieve the confirmation required for catalysis.
• The enzyme stalls, end of newly synthesized strand separate from
template and is directed to β’-5’ exonuclease.
• Bacteria also possess mismatch repair which operates after
replication
37. Rate of replication
• The single molecule of DNA that is the E. coli genome
contains 4.7 x 106 nucleotide pairs
• Replication of entire bacterial chromosome happens in ~40 min
at 37ºC i.e
• Each replication fork moves about 1000 nucleotides per second.
• A new round of replication can begin before the previous round
has been completed.
• The average human chromosome contains 150 x 106
nucleotide pairs which are copied at about 50 base pairs
per second per fork
38.
39. DNA replication in Eukaryotes
• Eukaryotes replicate their genome in small portions- replicons
• Replicon has its own origin from where replication fork
proceeds outward in both direction.
• In yeast- starts at ARS- autonomous replicating sequences-
conserved sequence of 11 bp.
• ARS is the binding site for multiprotein complex called ORC-
origin recognition complex.
40. • ORC (heteromeric protein) is described as molecular landing
pad- role in binding other proteins.
• ORC is bound throughout the cell cycle
• Early in G1 phase Proteins bind to ORC to assemble a protein –
DNA complex called pre-replication complex
• One of the principal proteins - Cdc6p (the replication
activator protein encoded by the yeast cdc6 gene)
41. • Then replication licensing factors (RLF) bind to initiate replication
• Two RLFs required: RLF-B and RLF-M.
• RLF-B is confined to the cytosol and has access to the
chromosomes only when the nuclear envelope disappears
early in mitosis- is present at the beginning of G1
42. • RLF-M is a heteromeric complex of the MCM proteins
(Mini chromosome maintenance proteins)
• Mcm proteins of LF loaded at origin at the late state of mitosis
associate into a ring shaped complex having helicase activity.
• These protein-protein interactions establish the pre-RC,
which consists of ORC, Cdc6p, the MCM complex, and
other proteins.
• Just before S phase, activation of protein kinases lead to the
activation of Mcm helicase and initiation of replication.
43. • At this point, two protein kinases act upon the pre-RC to
directly trigger DNA replication.
• One of these protein kinases is a complex of cyclin-
dependent protein kinase (CDK) and cyclin B, called cyclin
B-CDK.
• B-Cyclins accumulate at high levels just before S phase.)
• Cyclin B-CDK can phosphorylate sites in ORC, Cdc6p, and
several MCM subunits.
• Phosphorylation of Cdc6p causes it to dissociate from ORC,
whereupon it is degraded.
• Some of the MCM also dissociates
44. • Cyclin B-CDK also phosphorylates Cdc7p-Dbf4p, the
other protein kinase essential to activation of DNA
replication.
• Cdc7p interacts with ORC and Dbf4p interacts with the
replicator; together, Cdc7p-Dbf4p phosphorylates the
MCM complex.
• The consequence of these actions brings the cell into S
phase.
45. • These phosphorylation events serve as a replication switch
because once proteins in the pre-RC are phosphorylated,
the post-RC state is achieved.
• The post-RC state is incapable of re-initiating DNA
replication.
• This transformation ensures that eukaryotic DNA
replication occurs once, and only once, per cell cycle
46. Model for Initiation of the DNA Replication Cycle in
Eukaryotes (Yeast)
ORC=origin recognition complex
-is bound to replicators throughout
the cell cycle
Cdc6p -replication activator
protein
MCM- mini-chromosome
maintenance- a “replication
licensing factor (RLF)- permits
replication to occur
-Phosphorylation by these proteins
triggers DNA replication
47. • DNA Polymerase α
• -involved in initiation
• -synthesizes an RNA primer then adds dNTPs
• a complex of four subunits
• -50-kD and 60-kD are primase subunits;180-kD subunit DNA
polymerase
• -synthesizes 8-10 nt RNA primers, then adds DNA to the RNA
primers
• -low processivity of DNA synthesis (200 nt)
• -has no β’ -5’ exonuclease activity (proofreading), yet has high
fidelity
Eukaryotic DNA Polymerases
48. • DNA Polymerase
• -role in DεA repair (doesn’t participate in replication)
• DNA Polymerase
• -the DNA-replicating enzyme of mitochondria
49. • DNA Polymerase
• -the principal DNA polymerase in eukaryotic DNA replication
• -has β’-5’ exonuclease activity
• -consists of a 125 kD and a ~50 kD subunit
• -the 50 kd subunit interacts with PCNA (Proliferating Cell
Nuclear Antigen)
• -is highly processive when in association with PCNA
50. • DNA Polymerase
• -required for replication, but its role is unclear
• -may substitute for DNA polymerase d in lagging strand
synthesis
51.
52. Additional Proteins Involved in
Eukaryotic DNA Synthesis
• PCNA (Proliferating Cell Nuclear Antigen)
• -confers high processivity to DNA Polymerase
• -eukaryotic counterpart of the 2 Sliding Clamp of E. coli
• -PCNA also encircles the double helix, is a homotrimer of 37 kD
subunits
• RPA (Replication Protein A)
• -ssDNA-binding protein that facilitates the unwinding of the helix to
create two replication forks
• -the eukaryotic counterpart of the SSB protein of E. coli
• RFC (Replication Factor C)
• -the eukaryotic counterpart of the complex Clamp Loader of E. coli
53. • Leading strand synthesis
• 1) starts with the primase activity of DNA Pol- α to lay down a primer
• 2) then the DNA pol component of Pol α adds a stretch of DNA
• 3) RFC (Replication Factor C) assembles PCNA (Proliferating Cell
Nuclear Antigen) at the end of the primer
• 4) PCNA displaces DNA Pol α.
• 5) DNA polymerase binds to PCεA at the β’ ends of the growing to
carry out highly processive DNA synthesis –Polymerase switching
54. • Lagging strand synthesis
• 1) RNA primers synthesized by DNA polymerase α every 50 nt
and consist of 10-nt RNA + 10-20-nt DNA
• 2) polymerase switching as before to extend the RNA-DNA
primers to generate Okazaki fragments
• 3) when the DNA Pol approaches the RNA primer of the
downstream Okazaki fragment, RNase H1 removes all but the
last RNA nucleotide of the RNA primer
• 4) the FEN1/RTH1 exonuclease complex removes the last RNA
nucleotide
• 5) DNA Pol fills in the gap as the RNA primer is being removed
• 6) DNA ligase joins the Okazaki fragment to the growing strand
59. Telomeres
• The ends of eukaryotic chromosomes are called
telomeres (chromosomes are linear dsDNA
molecules).
• Telomere consists of a long series of short, tandemly
repeated sequences.
• General form Cn(A/T)m, where n>1 and m is 1-4.
• Telomeres are needed for chromosomal integrity
and stability (protect ends from degradation).
• The ends of the lagging strands cannot be copied
completely
60. • Telomerase was discovered by Carol W.
Greider and Elizabeth Blackburn in 1984 in
the ciliate Tetrahymena. Together with Jack W.
Szostak,
• Greider and Blackburn were awarded the 2009
Nobel Prize in Physiology or Medicine for
their discovery.
61. What about the ends (or telomeres) 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.
62. • In the absence of special telomere maintenance
• mechanisms, linear chromosomes shorten
progressively with every round of DNA
replication, eventually leading to cellular
senescence or apoptosis
69. Telomerase
• Maintains telomere length by restoring telomeres to the 3’-
ends of chromosomes.
• A ribonucleoprotein complex
• Consistists of a 126 kDal RNA-dependent DNA polymerase,
other proteins and a 450-nt RNA
• The telomerase polymerase is a “reverse transcriptase”
• The template sequence comes from the telomerase RNA
and is AAAACCCC
• Uses the 3’-end of the DNA as a primer and adds successive
repeats to it (TTTTGGGG for Oxytricia; TTTAGG for
humans).
70. • Telmerase contains a short RNA component, which
provided template for synthesis of repeats
• Telomerase uses the 3’OH of the G+T telomeric
strand as the primer for synthesis of tandem
TTGGGG repeats.
• The template RNA is positioned on DNA primer,
several repeats are added.
• After synthesis of TxGy strand by telomerase, the
complementary strand is synthesized by cellular
DNA pol.
71. • Then the enzyme translocate to begin the synthesis
again.
• The single stranded region is protected by specific
binding proteins in lower eukaryotes.
• In higher eukaryotes, the single stranded end is
sequestered in a specialized structure called T-
loop.
• The single stranded end is looped back and paired
with its complement in the ds portion of the
telomere.
72. Facts about Telomeres
• Somatic cells lack telomerase activity because the
telomerase reverse transcriptase (TERT), gene is switched
off
• Therefore, the telomeres get shorter with each cell division.
(About 50 bases are lost from each telomere every time a
normal cell divides.)
• Mammalian cells in culture will divide only ~ 50X
• “Telomere theory of aging”—cells senesce and die when
the telomeres are gone.
• Evidence?: Over-expression of telomerase activity extends
the life span of cells.
• Reactivation of Telomerase activity in cancer cells
74. Telomeres
• The ends of eukaryotic chromosomes are called
telomeres (chromosomes are linear dsDNA
molecules).
• Telomere consists of a long series of short, tandemly
repeated sequences.
• General form Cn(A/T)m, where n>1 and m is 1-4.
• Telomeres are needed for chromosomal integrity
and stability (protect ends from degradation).
• The ends of the lagging strands cannot be copied
completely
75. • In the absence of special telomere maintenance
• mechanisms, linear chromosomes shorten
progressively with every round of DNA
replication, eventually leading to cellular
senescence or apoptosis
76. • Telomerase was discovered by Carol W.
Greider and Elizabeth Blackburn in 1984 in
the ciliate Tetrahymena. Together with Jack W.
Szostak,
• Greider and Blackburn were awarded the 2009
Nobel Prize in Physiology or Medicine for
their discovery.
77.
78.
79.
80.
81. Telomerase
• Maintains telomere length by restoring telomeres to the 3’-ends of
chromosomes.
• A ribonucleoprotein complex
• Consistists of a 126 kDal RNA-dependent DNA polymerase, other
proteins and a 450-nt RNA
• The telomerase polymerase is a “reverse transcriptase”
• The template sequence comes from the telomerase RNA and is
AAAACCCC
• Uses the 3’-end of the DNA as a primer and adds successive repeats to
it (TTTTGGGG for Oxytricha; TTTAGG for humans).
82. • Facts about Telomeres
• Somatic cells lack telomerase activity because the
telomerase reverse transcriptase (TERT),
gene is switched off
• Therefore, the telomeres get shorter with each cell
division. (About 50 bases are lost from each telomere
every time a normal cell divides.)
• Mammalian cells in culture will divide only ~ 50X
• “Telomere theory of aging”—cells senesce and die when
the telomeres are gone.
• Evidence?: Over-expression of telomerase activity
extends the life span of cells.
• Reactivation of Telomerase activity in cancer cells
84. • Telmerase contains a short RNA component, which provided
template for synthesis of repeats
• Telomerase uses the 3’OH of the G+T telomeric strand as the
primer for synthesis of tandem TTGGGG repeats.
• The template RNA is positioned on DNA primer, several repeats
are added.
• After synthesis of TxGy strand by telomerase, the
complementary strand is synthesized by cellular DNA pol.
• Then the enzyme translocate to begin the synthesis again.
• The single stranded region is protected by specific binding
proteins in lower eukaryotes.
• In higher eukaryotes, the single stranded end is sequestered in a
specialized structure called T-loop.
• The single stranded end is looped back and paired with its
complement in the ds portion of the telomere.
85. Rolling circle model of DNA replication
(3.11):
1. Common in several bacteriophages
including .
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.
86. • Control of Replication
• With their multiple origins, how does the eukaryotic cell know which origins have been already replicated
and which still await replication?
• An observation: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2
nucleus does not begin replicating again even though replication is proceeding normally in the S-phase
nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.
• Two control mechanisms have been identified — one positive and one negative. This redundancy probably
reflects the crucial importance of precise replication to the integrity of the genome.
• Licensing: positive control of replication
• In order to be replicated, each origin of replication must be bound by:
• an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
• Accessory proteins called licensing factors. These accumulate in the nucleus during G1 of the cell cycle.
They include:
– Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
– MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.
• Once replication begins in S phase,
• Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes).
• The MCM proteins leave in front of the advancing replication fork.
• Geminin: negative control of replication
• G2 nuclei also contain at least one protein — called geminin — that prevents assembly of MCM proteins
on freshly-synthesized DNA (probably by blocking the actions of Cdt1).
• As the cell completes mitosis, geminin is degraded so the DNA of the two daughter cells will be able to
respond to licensing factors and be able to replicate their DNA at the next S phase.