Class notes
Class notes Biomedical Sciences (BSc) (BB2704) Essential Cell Biology, ISBN: 9780393680393
- Course
- Institution
- Book
Lecture Notes BB2704 Molecular and Cellular Biology at Brunel University
[Show more]
Connected book
Book Title:
Author(s):
- Edition:
- ISBN:
- Edition:
More summaries for
-
Essential Cell Biology 5th Edition by Alberts
-
Essential Cell Biology 5th Edition by Alberts
-
COMPLETE TEST BANK: ESSENTIAL CELL BIOLOGY 5TH EDITION ALBERTS [ CONTAIN ANSWER KEY Questions with 100% correct Answer.
Seller
Follow
eunicebediako_
Reviews received
Content preview
BB2704 Cellular and Molecular BiologyDNA replication
- DNA replication is necessary to generate genetic material for the daughter cells. DNA
replication is the copying of double-stranded DNA. It is necessary for cell reproduction
before cell division. There are 5 phases in the cell cycle:
o G1 = growth phase 1
o G0 = growth arrest
o S = synthesis phase (DNA replication)
o G2 = growth phase 2
o M = mitosis (cell division)
- DNA synthesis is the molecular events that allows assembly of new DNA strands
- DNA replication is a more complex term including:
o DNA chain synthesis
o Initiation, elongation and termination
o Accuracy
o Daughter strand separation
o Distribution of chromosomes
- Watson and Crick proposed that DNA replicated semi-conservatively. The semi-conservative
model is the intuitively appealing model, because separation of the daughter strands
provides two templates, each of which carries all the information of the original molecules
- The three models:
o Semi-conservative model
Two parental strands separate and each makes a copy of itself. After one
round of replication, the two daughter molecules each comprises of one old
and one new. After two rounds, two of the DNA molecules consist only of
the new material, while the other two contain one old and one new strand.
o Conservative model
The parental molecule directs synthesis of an entirely new double-stranded
molecule. After one round of replication, one molecule is conserved as two
old strands
o Dispersive model
Material in the two parental strands is distributed more or less randomly
between two daughter molecules
- In 1958, Meselson and Stahl devised an experiment to determine whether DNA replicated
following a conservative, semi-conservative or dispersive model.
- The experiment includes:
o E. coli in a medium with the heavier 15N isotope
o E. coli cells with only 15N in their DNA were transferred to a 14N medium and were
allowed to divide
o DNA was extracted periodically and was centrifuged
o Each sample was centrifuged in a caesium chloride solution
o The heavier the DNA, the further it moved down. Mixed 14N and 15N DNA is
intermediate in mass between the two.
o The original 15DNA moved to the lowest position
o After one generation, all the DNA moved to an intermediate position, indicating the
presence of only mixed. This was because the DNA of this generation contained one
strand of the parent molecule and one new strand. This is consistent with both semi-
conservative and dispersive models.
, o If it was the conservative model, one DNA would have been heavy and the other
light
o In the second generation, half the DNA was intermediate and half was light. This is
consistent with only the semi-conservative model.
How does replication start?
- Origin of replication: A unique DNA sequence at which DNA replication is initiated and
proceeds bi-directionally. The point at which replication occurs is called the replication fork.
- In a circular DNA molecule (bacteria), there is a single ORI that spreads bi-directionally
- In a linear DNA molecule (chromosome), there are multiple ORI that spread bi-directionally
along the chromosome, away from the ORI
- The ORI in E. coli is called OriC, which is only 245bp in length. It consists of:
o AT-rich region
o DnaA boxes
o GATC methylation sites
AT-rich core only has 2 hydrogen bonds between A and T, therefore they are easier to break
than GC-bonds
- DNA replication is initiated by the binding of the DnaA proteins to the DnaA box sequences.
- Monomers of the replication initiator protein DnaA bind to the 9 mer repeats, and the DNA
coils around this protein complex forming a protein core. This leads to the separation of DNA
strands. Additional proteins, DnaB/DnaC, join the complex forming replication forks.
Eukaryotes
- In 1992, Bell and Stillman isolated a yeast protein that binds to ARS (autonomously
replicating sequence, which allows plasmid to replicate autonomously). This protein is called
origin of recognition complex (ORC)
- DNA sequences at the replication origins have been harder to isolate. However evidence
suggests that the origin of replication in mammalian cells is similar to yeast type origins.
- All the origins form a base for assembly of a group of proteins known collectively as the pre-
replication complex (pre-RC):
1. The origin DNA (ARS) is bound by ORC (six different protein subunits)
2. Two cell division cycle proteins (Cdc6 and Cdt1) bind to the ORC
3. Cdc6 and Cdt1 load the mini chromosome maintenance (MCM) protein complex
- This complex of proteins indicates that the replication origin is ready for activation. This
process by which pre-RC is assembled is called licensing. A mutation in a protein can cause
replication not to initiate.
Replication factories
- The replication proteins are clustered together in particular locations in the cell and may be
regarded as a “Replication Factory” that manufactures DNA copies. The DNA to be copied is
fed through the factory. There may be up to 100,000 replication origins in the mammalian
nucleus. These foci are not permanent and only found in the S-phase.
- DNA replication has been shown to proceed in a bidirectional fashion
- From a single origin, two replication forks can emerge and proceed in opposite directions.
- DNA replication in E. coli takes about 40 minutes. This is because of its naked DNA.
Replication speed → 50,000bp/min
- DNA replication in humans takes about 8 hours. This is because of its compact DNA.
Replication speed → 2000bp/min
,Organisation of DNA replication fork
- The replication fork is created by helicases, which break the hydrogen bonds holding the two
DNA strands together. The resulting structure has two single strands which serve as
templates.
- DNA is always synthesised in the 5’ to 3’ direction. The chemical polarity of a dsDNA
molecule is antiparallel. One strand goes from 5’ to 3’ while the other goes from 3’ to 5’. This
presents problems for an organism replicating its DNA.
- One strand in the replication fork is synthesised as a continuous molecule, but the other is
made in short sections or pieces. These strands are referred to as leading (continuous) and
lagging (discontinuous) strands. Therefore the replication fork is a semi-discontinuous model
- The leading strand is being synthesised in the same direction as the growing replication fork.
A polymerase ‘reads’ the leading strand template and adds complementary nucleotides on a
continuous basis.
- The lagging strand’s direction of synthesis is opposite to the direction of the growing
replication fork. Because of its orientation, replication of the lagging strand is more
complicated. The lagging strand is synthesised in short, separated segments (Okazaki
fragments). A primase ‘reads’ the template DNA and initiates synthesis of a short
complementary RNA primer. A DNA polymerase extends the primed segments forming the
fragments.
Termination of replication
- Termination of replication is one of the least well understood areas of DNA biology.
- The termination sequences in E. coli are called ter sites, which serve as binding sites for the
Tus terminator protein. Tus can block the progression of replication forks in specific
directions.
- Replication terminates when the two growing forks meet in the terminus region. In E.coli,
the 5 ter sites, J, G, F, B and C are arranged opposed to ter sites H, I, E, D and A, and can
arrest the fork progressing in the clockwise direction and can block the anticlockwise
direction, respectively.
- The replication fork progressing in a clockwise direction will encounter the terC site first and
pause. If the fork progressing from the anticlockwise direction meets the clockwise fork
while paused, replication is terminated. However if it does not meet its anti-fork it will
proceed until it reaches the next termination site, terB, where it will pause again, etc.
- Multiple ter sites are important as backups, to ensure that termination is completed.
Multiple regions to entrap the replication fork also means that if an inactivating mutation
arises within a ter site, then arrest can still occur at another ter sequence
- The E.coli protein that is responsible for termination is a 36kDa protein names Tus (Terminus
Utilisation Substance) that binds 23bp ter sites and arrests the replication helicase, DnaB,
responsible for separating the two strands of DNA
- The Tus-Ter complex is known to terminate replication by arresting the replication
machinery in a polar manner
- Little is known about termination of replication in eukaryotes. Replication forks possibly
meet at random positions and termination involves ligation of the ends of the new
polynucleotides
- Telomeres are specialised structures at the ends of linear chromosomes. Their function is to
protect the end of the chromosome from fusion with neighbouring chromosomes. They
consists of repeat arrays of the sequence (TTAGGG) n. In humans telomeres can be 20-40kb in
length.
, How do the new strands of DNA get made?
- DNA polymerases catalyze the formation of DNA through the addition of nucleotides to the
growing strand of new DNA. All prokaryotic and eukaryotic DNA polymerases add
nucleotides to the 3′-OH end of a polynucleotide chain, so that the new chain grows in the 5’
3’ direction. The precursor for DNA synthesis is a nucleoside triphosphate, which loses
the terminal two phosphate groups in the reaction.
1. Incoming nucleotide
2. Nucleophilic attack on phosphate
3. Formation of phosphodiester bond
4. Pyrophosphate formed as by-product
To initiate this reaction, DNA polymerases require a primer with a free 3 ′-hydroxyl group
already base-paired to the template.
DNA Polymerase
- There are total of 5 different DNA polymerases that have been discovered in E.coli
o DNA Polymerase I: major repair enzyme
o DNA Polymerase II: replication restart
o DNA Polymerase III: principal DNA replication enzyme (replicase)
o DNA Polymerase IV: functions in repair
o DNA Polymerase V: functions in repair
- Eukaryotes have three different DNA Polymerases
o DNA polymerase , and : main polymerases involved in DNA replication
(Replicases)
o DNA polymerase : DNA repair
o DNA polymerase : mitochondrial DNA replication
- DNA polymerase also has exonuclease activities:
o Some polymerases have the ability to destroy newly synthesised strands right after
the new strand has been made. It can do so in both directions (5’ → 3’ and 3’ → 5’).
This mechanism is important for the elimination of the primer.
o The 3’-5’ exonuclease activity acts as a proof reading mechanism: correcting
mistakes made by the polymerase. It removes incorrectly matched bases, so that the
polymerase can try again.
- DNA polymerase moves along a single strand of DNA, building the complementary strand as
it goes. The two stranded molecule passes through the DNA polymerase molecules after
synthesis is complete. If the wrong base is inserted then the bond is unstable. Because the
double strand is passing through the DNA polymerase the missing base can be detected and
replaced.
- The growing nucleotide chain on the synthesis domain occasionally leaves the polymerase
site of DNA polymerase I and migrates to the exonuclease site where the incorrect base pair
is cleaved off
- DNA polymerases have a common structure, also referred to as
The Klenow fragment. It approximates the shape of a right
hand with domains that are referred to as the fingers, the
thumb and the palm. The fingers place the DNA in the right
position. The thumb binds with the DNA. The palm is the active
site of the enzyme. The Klenow fragment includes a domain
with 3’ → 5’ exonuclease activity that participates in
proofreading.
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller eunicebediako_. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $7.75. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
67866 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now
Recently viewed by you
Exam (elaborations) ·
Mobility ATI Questions and Answers Already Passed
Exam (elaborations) ·
NEHA Exam| 303 Questions| With Complete Solutions
