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Cell Cycle: Events that occur in a single round

of cell division
The mitotic cell cycle can be divided into 4
phases: G1, S, G2 and M.
DNA synthesis
1. DNA synthesis occurs during the S phase
occurs resulting
resulting in the duplication of chromosome
duplication of
2. Chromosome segregation occurs during

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ve
of igh re
chromosome
mitosis or the M phase.

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le
En

s
3. G1 and G2 are gap phases allow the cells to

nt
ie
prepare for the next events in cell cycle

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Example: Many eukaryotic cells use G1 phase
of the cell cycle establishes high level of
nutrients to allow the completion of cell
division. s om Pr e
o Ce pare
Gap phases serves two purposes: The gap om io ll d f
r t ivis or
period provides a) time for the cell to prepare Ch e ga ion
for the next phase of the cell cycle,( synthesis e egr
S
of proteins, and nutrients). If there is a n
problem, the cell cycle check points arrest the
cell cycle until the defects are rectified or
provide the cell with necessary time to
complete the step appropriately 2) check the
previous phase of the cell cycle completed
appropriately.
Formation of Pre Replicative Complex

Initiator proteins are called Dna A


in E.coli, T antigen in SV40, ORC in
Replicator is a sequence. ORC , a
yeast/ mammals)
protein binds replicator.
Proteins, Cdc6 and Cdt1, load the
Helicases (Dna B and its helper
helicase Mcm 2-7 in eukaryotes
Dnac in prokaryotes) ; Mcm
which is comparable to DnaB in
complex in eukaryotes
function in prokaryotes.
Formation of pre replicative complex does not lead to the immediate
unwinding of DNA or the recruitment of DNA polymerase.
1. Pre RC are formed in G phase has to be activated . It requires two proteins
kinases: cdk and Ddk.
2. These kinases are inactive in G1 phase and are active in S phase.
3. Once active, these protein kinases phosphorylate the replication proteins
Pre RC Formation and regulation of its activation allows only a single
round of replication during each cell cycle. Pre RC formed during G1
phase are activated to initiate replication only when cells move from
G1 to S phase.
Recall in bacteria DNA replication is regulated by Dna A-ATP and seq
A levels so as to enable the chromosome to replicate once during
each cell division.
DNA polymerase moves along the template strand in a 3'-5' direction, and the
daughter strand is formed in a 5'-3' direction.
This difference enables the resultant double-strand DNA formed to be composed
of two DNA strands that are antiparallel to each other.
The 3'-5' exonuclease activity of the enzyme allows the incorrect base pair to be
excised (this activity is known as proofreading). Following base excision, the
polymerase can re-insert the correct base and replication can continue forwards.
This preserves the integrity of the original DNA strand that is passed onto the
daughter cells.
Nick translation
Step1. Formation of pre replicative complex in G1
phase
Step 2: Activation of Pre replication complex in S
phase by Cdk and and Ddk
Step3. DNA unwinding at the origin by Mcm 2-7
Step 4. Recruitment of a number of auxillary
replication factors and polymerases like and .
Step 5. Polymerase and primase are recruited
after the joining of and polymerases.
Step 6: Pol /primase complex synthesizes an RNA
primer and briefly extends it.
The resulting primer-template junction is recognized
by the eukaryotic sliding clamp (PCNA) and
clamp loader (RF-C) .The clamp loader assembles
the sliding clamp.
Step 7. . Either DNA pol or recognize the primer
and begins leading strand synthesis.
Step 8: After a period of DNA unwinding, Pol
/primase synthesizes additional primers , which
allow the initiation of lagging strand synthesis.

Pol and work perhaps separately one for leading


strand and other for lagging strand synthesis.
Because of its 5 to 3 exonuclease activity, DNA polymeraseI removes RNA
primers and fills the gaps between okazaki fragments with DNA. The
resultant DNA fragments can then be joined by DNA ligase.
Lagging strand synthesis is unable to copy the extreme ends of DNA on linear
chromosomes.
Cellular enzymes FEN1 (flap endonuclease
1) and RNase H remove RNA primers at
the start of each leading strand and at the
start of each Okazaki fragment, leaving
gaps of unreplicated template DNA. Once
the primers are removed, a free-floating
DNA polymerase I lands at the 3' end of
the preceding DNA fragment and extends
the DNA over the gap. However, this
creates new nicks (unconnected sugar-
phosphate backbone).

In the final stage of DNA replication, the


ligase joins the sugar-phosphate
backbones at each nick site. After ligase
has connected all nicks, the new strand is
one long continuous DNA strand, and the
daughter DNA molecule is complete.

Lagging strand synthesis is unable to copy the extreme ends of linear chromosomes which carries
an RNA primer. Once the primer is removed the DNA polymerase cannot copy the template end
as the DNA polymerase works in 5-3 direction. (To better understand the importance of
telomerase draw a DNA with 53 ends and its replication
Telomerase- Imp features NOVEL DNA POLYMERASE THAT
DOES NOT REQUIRE AN EXOGENOUS TEMPLATE

It is a ribonucleoprotein comprising a DNA polymerase protein and


RNA strand.

Like all other DNA polymerases it uses the 3 OH group

However , it is unlike DNA pol. It does not require any template

4. The RNA component of telomerase serves as the template for


adding telomeric sequence to the 3 end of chromosome. Telomerase
extends 3-OH of ssDNA using its RNA template.

5. The polymerase activity of telomerase is comparable to reverse


transcriptase, an RNA-dependent DNA polymerase but not DNA
dependent DNA polymerase
Although telomerase only directly extends
the 3' end of the telomere, by providing an
additional template for lagging strand DNA
synthesis, both ends of the chromosome
are extended.
Telomerase: RNA-protein complex that elongates
the lagging strand template from its 3 OH end .
1. The sequence of RNA component of the
telomerase is partly complimentary to the
telomeric sequence (3 OH region of the
template)
2. In humans, the telomeric RNA sequence is 5
TAACCCTAA-3 . This strand anneals to the
single strand DNA at the 3 end.
3. Annealing occurs in such a way that primer
template junction is formed and the polymerase
activity of telomerase can act now to extend the
chain at 3 end.
4. The polymerase activity of telomerase is
comparable to reverse transcriptase, an RNA-
dependent DNA polymerase but not DNA
dependent DNA polymerase
5. In the case of fig, after the addition of last 3
nucleotides on the 3 end of DNA that is
complementary to RNA, telomerase disengages
from the DNA product and reanneals to the last
three nucleotides. The process of elongation is
First Recombinant DNA molecule
Terminal Transferase:
Tailing method Stanley Cohen- Annie Chang
DNA polymerase
Paul berg template independent
and adds nt at 3OH
Different steps in the
preparation of cDNAs and
their cloning.

Reverse transcriptase:
RNA dependent DNA
polymerase
Taq Polymerase : DNA dependent DNA polymerase. Isolated from Thermus aquaticus,
grows in hot springs. Used in Polymerase Chain Reaction ( PCR) that amplifies DNA in
test tube rxns . Requires dNTPs (dTTP, dGTP, dATP, dCTP + primers + Temp

No of DNA
Cycle molecule

PCR amplifications are used to detect bacterial and viral infections


PCR amplification is used for sex determination in prenatal cells
PCR is used in Molecular evolution
Overall it is a technical revolution in molecular genetics
Dideoxy DNA sequencing- Sangers method

Prepare 23 dideoxy nucleotides of all the 4 bases. ddATP,


ddTTP, dd CTP and ddGTP. These molecules can be
incorporated into DNA by E.coli DNA polymerase because
they have a normal triphosphate; however once
incorporated into a growing strand, the ddNTP cannot form
a phosphodiester bond with the next incoming dNTP.
Growth of DNA stops when ddNTP is incorporated.

Sanger sequencing reaction consists of i) a DNA strand to


be sequenced, ii) a short labeled piece of DNA, iii) the
primer that is complimentary to the end of that DNA
strand,iv) a carefully controlled ratio of one particular
ddNTP with its normal dNTPand the other three dNTPS.

In the presence of DNA polymerase, normal polymerization


will begin from the primer, when a ddNTP is incorporated ,
the growth of that chain will stop. If the correct ratio of
ddntp:dNTP is chosen a series of labelled strands will
result, the lengths of which are dependent on the location
of a particular base relative to the end of the DNA.
Resultant labelled fragments are sepaated by size on
polyacrylamide gels. Autoradiography is then performed.
Important questions
Bacterial DNA polymerases
Polymerase III ( subunit composition ) and activities
Origin of replication and proteins that bind origin in prokaryotes and eukaryotes
Eukaryotic DNA polymerases
How do you determine 5-3 helicase activity?
How do you determine polymerase activity and nuclease activity associated with
polymerase I or III ?
What is 5-3 nuclease or 3-5 exonuclease activities/What is their importance
What is Tail and Head growth?
What is the chemistry of DNA synthesis? Where does the energy come form?
What are the proteins and enzymes in bacterial DNA replication? Their functions
Why RNA primers are required?
What is the difference between PCR and cellular DNA replication?
Describe 3-4 polymerases and their applications if any?
What is nick translation?
What is a telomerase/ How does it work?
What is the function and importance of seq A protein?
Describe step reaction of a bacterial ligase
Describe Cell cycle and most important events that happen at different stages?
Pre RC formation and its activation in eukaryotes
Pol polymerase and primase activities
This unique enzyme has two distinct polymerase activities: a 5-3 DNA-dependent
DNA polymerase, and a 5- 3 DNA-dependent RNA polymerase.
The RNA polymerase activity is a primase. Because of this, the enzyme is often
referred to as Pol :primase. It is the only enzyme known to have both DNA
polymerase and primase activities, and the only one capable of self-primed DNA
synthesis on a previously unprimed ssDNA.
Pol :primase is a heterotetramer:
180 kDa DNA pol catalytic subunit
68 kDa - structural; protein-protein interactions
48 kDa -- primase catalytic subunit
55 kDa primase subunit; bridge with DNA pol
(protein subunit sizes from human cells)

(Recall that prokaryotes use primers that consist of RNA only, synthesized by
DnaG primase.)

Eukaryotic pols can generally use DNA or RNA primers. But eukaryotic clamp
loaders generally prefer DNA primers.
Pol is another multisubunit nuclear enzyme. In Homo sapiens, it is composed of 4
subunits:
125 kDa-- pol catalytic subunit; 66 kDa-- interaction with processivity factor;
multimerization (?); 50 kDa-- structural; protein-protein interactions; 12 kDa-- structural;
protein-protein interactions

he catalytic subunit has 5- 3 DNA polymerase activity and an intrinsic 3-5' exonuclease
activity. The smaller subunits appear to be involved in holding the multisubunit structure
together (via protein-protein interactions). Addition of the 66 kDa subunit appears to
result in dimerization of the tetrameric complex, so Pol , like Pol III, may also be a
dimeric replicating machine.

Pol requires an associated 30 kDa protein, called proliferating cell nuclear


antigen (PCNA), for full polymerase activity and processivity.
Functional PCNA is a homotrimer (120 kDa) and acts as the processivity factor.
The 66 kDa subunit is needed for interaction between Pol d and PCNA.
So unlike the situation in Pol III, the clamp does not interact directly with the catalytic
subunit.
PCNA functions as a sliding clamp to increase the processivity of Pol up to 50-
fold.
PCNA requires a clamp loader to become associated with the template primer.
The matchmaker in this case is an associated protein complex known as replication
factor C (RFC), a heteropentamer, and together with ATP and PCNA it is loaded onto
the template. Pol is a typical replicase complex, similar to bacterial Pol III holoenzyme
DNA polymerase
Pol is another multisubunit nuclear DNA polymerase. The human pol has 4
subunits:
260 kDa-- pol catalytic subunit
59 kDa-- multimerization (?)
17 kDa-- structural; protein-protein interactions
12 kDa-- structural; protein-protein interactions
The Pol e catalytic subunit is one of the largest polymerase activities yet described. It
also has
3-5' exonuclease activity. The N-terminal portion contains the catalytically important
residues.
The C-terminal part consists of a 1,000 amino acid extension that is unique to Pol.
This domain is known to interact with regulatory proteins, some of which are involved
in cell cycle regulation (check point control).

Because of its high processivity and proofreading activity, it has been


further suggested that Pol is involved in primer elongation, in addition
to Pol . In support of this, Pol has been detected at the replication fork (along with
Pol and Pol ) in mammalian cell extracts. Biochemical and genetic studies in yeast
suggest that Pol is also involved in some DNA repair synthesis.
DNA polymerase
smallest and simplest of the classical eukaryotic polymerases; composed of a single
~40-48 kDa protein. Pol is not highly active and is not very processive. It has no
intrinsic exonuclease activities. Its preferred template is duplex DNA with short gaps,.