Dna replication prokaryotes

DNA REPLICATION IN
PROKARYOTES
DR. ANU P. ABHIMANNUE,
ASSISTANT PROFESSOR,
DEPARTMENT OF BIOTECHNOLOGY,
ST. MARY’S COLLEGE, THRISSUR

REPLICATION
Prokaryotic DNA Replication
is the process by which a
prokaryote duplicates its DNA
into another copy that is
passed on to daughter cells
DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR. 2

SCHEMES OF REPLICATION
DR ANU P. A., ST. MARY'S COLLEGE, THRISSUR.
3
Three types of DNA replication methods were postulated
– Conservative method
– Semi- Conservative method
– Dispersive method

CONSERVATIVE METHOD
• In conservative replication, the original strands would
remain together as would the two newly synthesized
strands.
• Hence, one of the daughter duplexes would contain only
parental DNA, while the other contain only newly
synthesized DNA.
4

SEMI-CONSERVATIVE METHOD
• Semi-conservative mode was suggested by Watson and Crick in 1953.
• The daughter duplexes consist of one complete strand inherited from the
parental duplex and one complete strand that has been newly synthesized.
• It is said to be semiconservative because each daughter duplex contains one
strand from the parent structure.

DISPERSIVE METHOD
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• The parental strands would be broken into fragments, and
new strands synthesized in short segments, which further
join together to form a complete strand.
• As a result, the daughter duplexes would contain strands that
were composites of old and new DNA.

POSSIBILITIES O DNA
REPLICATION
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EVIDENCE
• To gain evidence on DNA replication, Studies on bacteria was
conducted by Matthew Meselson and Franklin Stahl of the
California Institute of Technology in 1957.
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Franklin Stahl : Born October 8,
1929), American molecular
biologist and geneticist.
Matthew Meselson: Born May
24, 1930, American
geneticist & molecular biologist

PRINCIPLE
• They used heavy (15N) and light (14N) isotopes of nitrogen
to distinguish between parental and newly synthesized DNA
strands.
• The density of a DNA molecule is directly proportional to
the percentage of 15N or 14N atoms it contains.
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Procedure
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Bacteria was grown in medium containing 15N-
ammonium chloride (heavy isotope) as the sole
nitrogen source
These cultures were washed free of the old
medium and incubated in fresh medium
containing light, 14N and were analyzed at
increasing intervals over a period of several
generations
DNA was extracted from the
bacterial samples and subjected to
equilibrium density-gradient
centrifugation

https://www.mun.ca/biology/scarr/Meselson_StahL_experiment.html
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EQUILIBRIUM DENSITY-GRADIENT
CENTRIFUGATION
• Extracted DNA was mixed with a concentrated solution of CsCl and
centrifuged to equilibrium at high speed in an ultracentrifuge.
• Cesium ions form a density gradient with the lowest density of Cs at the
top of the tube and greatest concentration at the bottom.
• During centrifugation, DNA fragments within the tube become localized
at a position having a density equal to their own density, which in turn
depends on the ratio of 15N/14N.
• The greater the 14N content, they localize higher in the tube.
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CONCLUSION
• The appearance of a hybrid band and the disappearance of the
heavy band after one generation eliminates conservative
replication.
• The subsequent appearance of two bands, one light and one
hybrid, eliminates the dispersive scheme.
• As long as replication continued semi-conservatively, the original
heavy parental strands remain intact and occupied a smaller
percentage of the total DNA. Hence, DNA replication was found
to be semi-conservative in nature.
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PROKARYOTIC DNA REPLICATION
• In 1963, John Cairns reported
the process of replication in E.
coli bacteria by autoradiography.
• The replication process can be
broadly divided into 3 sections
– Initiation
– Elongation
– Termination
Cairns: (21 November 1922 – 12
November 2018); British physician and
molecular biologist

ORIGIN
• Replication begins at a specific site on the chromosome called origin.
• Origin of replication of E. coli is called oriC
• It is of 245 bp length, where a number of proteins bind for initiation.
• It is a conserved sequence in prokaryotes, rich in AT residues.
• Ori C consists of two types of sequences
– 13mer - three repeats of 13bp
– 9mer - five repeats of 9bp
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PROTEINS IN INITIATION
DnaA and DnaC : proteins helping in the recognition of the specific
sequence at oriC
Helicase: Enzyme, in presence of ATP unwinds DNA by breaking hydrogen
bonds between the nitrogenous base pairs to form replication forks .
Single-strand binding proteins: coat the single strands of DNA near the
replication fork to prevent them from winding back into double helix.
DNA gyrase: Type II topoisomerase enzyme, relieves the mechanical strain
that builds up during replication by removing positive supercoils
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HELICASE
• E. coli has 12 different helicases.
• One specific helicase ie, DnaB helicases serves as the major
unwinding enzyme during replication.
• DnaB helicase consists of six subunits arranged to form a ring-
shaped protein that encircles a single DNA strand.
• It is first loaded onto the DNA at the origin and translocates in a 5´
→ 3´ direction along the lagging-strand template, unwinding the
helix as it proceeds.

DNA GYRASE
It is a type II topoisomerase enzyme,
change the state of supercoiling in DNA
molecule.
Moves along the DNA ahead of replication
fork, removing positive supercoils.
Mechanism: Cleaves both strands of the
DNA, passing a segment of DNA through the
break to the other side, and then seals the
cuts. It require ATP hydrolysis

INITIATION
• Dna A protein with ATP, binds to the 9mer sequences of oriC.
• This binding allows the opening of 13mer sequences of oriC by bending
the DNA.
• Helicase bind to 13mer repeats which are recognized by DnaC.
• Once helicase is settled on oriC (at 13mer) the DnaC protein will be
released.
• Helicase unwinds the DNA duplex into a Y – shaped structure called
replication fork
• Meantime, tension developed is relieved by DNA gyrase

REPLICATION FORK
• The replication origin forms a Y shape,
and is called a replication fork.
• The two replication forks move
outwards in opposite directions that is,
bi directionally
• At the end they meet at a point across
the circle from the origin, where
replication is terminated.

ELONGATION

Enzymes
 DNA pol III – Major enzyme required for DNA synthesis.
 Primase - Synthesize a short segment of RNA called primer
24

DNA POLYMERASES
• Pioneer work in the study of DNA Pol
was carried out by Arthur Kornberg at
Washington University in the 1950s
• It adds nucleotides to the growing DNA
chain that is complementary to the
template strand.
• For polymerization to proceed, the
enzyme need DNA and energy from all
four dNTPs.
Arthur Kornberg (March 3, 1918 –
October 26, 2007) American
biochemist who won the Nobel Prize in
Physiology or Medicine 1959

TYPES OF DNA POL
• In 1969, studies on a mutant strain of E. coli revealed that apart from Kornberg
enzyme (DNA polymerase I), several distinct DNA polymerases are present.
• A typical bacteria contains 300 to 400 molecules of DNA polymerase I but only
about 10 copies of DNA polymerase III.
• In prokaryotes, three main types of polymerases are known:
– DNA pol I - accessory enzyme in DNA replication
– DNA pol II - along with DNA pol I, is required for DNA repair.
– DNA pol III – Major enzyme required for DNA synthesis.
26

DNA POLYMERASE I
DNA polymerase I is considered as three different enzymes
in one as it has 3 different functions.
1. 5´→ 3´ exonuclease activity can degrade RNA stretches
created by primase in lagging strand.
2. 5´→ 3´ polymerase activity that fills the resulting gap in
lagging strand
3. 3´→5ˊ exonuclease activity
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DNA POL STRUCTURE
The holoenzyme consist of 10 different
subunits
1. Two core polymerases which
replicate the DNA,
2. Two or more clamps, which allow the
polymerase to remain associated
with the DNA,
3. A clamp loading complex, which
loads each sliding clamp onto the
DNA.

DNA POL STRUCTURE - CLAMP
• Clamp – non - catalytic component of the holoenzyme for
keeping polymerase associated with the DNA template.
• Doughnut-shaped clamp encircles DNA and provides 2
contrasting properties for the enzyme:
1.long stretch association of enzyme with template
2.loose attachment with the template for easy
movement.
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DNA POL STRUCTURE – CLAMP
LOADER
• Clamp loader – multi subunit of enzyme containing two t subunits,
which hold the core polymerases
• Clamp loading complex in ATP-bound state assembles at primer-
template junction for holding the clamp.
• Once DNA moves through the opening in the clamp wall, ATP bound to
the clamp loader is hydrolyzed, causing the release of the clamp, which
closes around the DNA.
• The clamp is then ready to bind polymerase III.
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LIMITATIONS OF DNA POLYMERASE
• Two important restrictions:
– DNA Pol add Nucleotides only in the 5′ to 3′ direction.
– Enzyme requires a free 3′-OH group to add nucleotides,
so, if a free 3′-OH group is not available. Then how does
it add the first nucleotide?
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PRIMER
• A short segment of RNA can provide the required 3´ OH terminus
for DNA Pol and is called a primer.
• This 3´ OH carries out a nucleophilic attack on the 5´ phosphate of
the incoming Nucleoside TriPhosphate.
• Primer is five to ten nucleotides long and complementary to
template DNA.
• Primer is synthesized by RNA polymerase known as primase
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PRIMOSOME
• Primosome is a combination of
primase and helicase enzymes.
• Helicase moves along the lagging-
strand template processively (i.e.,
without being released from the
template strand during the
lifetime of the replication),
thereby opening the duplex.
• Primase periodically binds to the
helicase and synthesizes primers.

REPLISOME
• Replisome refers to the entire complex of proteins that are active
at the replication fork.
• Complex includes DNA polymerase III holoenzyme, the helicase,
SSBs and primase.
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LEADING STRAND
• Strand complementary to 3′ to
5′ parental DNA strand
• synthesized continuously in a
single stretch
• Synthesized towards the
replication fork
• Synthesized in 5′to3′ direction.
LAGGING STRAND
• Strand complementary to 5′ to 3′
parental DNA
• Synthesized discontinuously, in
fragments - Okazaki fragments.
• Synthesized away from the
replication fork.
• Synthesized in 3′ to 5′ direction

https://www.netclipart.com/isee/xmoTbT_dna-structure-clipart-dna-replication-enzymes-unzip-the/

OKAZAKI FRAGMENTS
• Short fragments on the lagging
strand is known as Okazaki
fragments.
• Discovered by Reiji Okazaki of
Nagoya University, Japan.
• Synthesis of Okazaki fragments
require individual primer.
October 8, 1930 – August 1, 1975,
Japanese molecular biologist

• After the synthesis of leading and lagging
strand, the polymerase is detached from the
site of replication.
• Thus bringing an end to elongation
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Termination

ENZYMES
 RNase H – An endogenous enzyme that cleaves the RNA strand of an
RNA–DNA duplex in lagging strand.
 DNA polymerase I – exonuclease activity, removing primers
 DNA ligase – Joining of DNA fragments
 DNA topoisomerase II – aids dissociation of 2 different circular DNA
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DNA LIGASE
• DNA ligase joins Okazaki fragments into a continuous strand.
• It facilitates the joining of DNA strands by catalyzing the
formation of a phosphodiester bond
• Catalyze 2 covalent phosphodiester bonds between 3‘OH end and
5' phosphate end of nucleotides.
• Two ATP molecules are consumed for each phosphodiester bond
formed.
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TERMINATION
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Multiple primers at lagging strand are cleaved by
RNase H
Primers removed by DNA polymerase I & fills
these gaps by the addition of nucleotides
DNA polymerase proofreads the sequence
for avoiding error in replication
Finally, the enzyme DNA ligase fills the gap

PROOF READING
• Incorrect nucleotides are often removed by DNA polymerase I
during termination of replication.
• The enzyme directs into the 3ˊ → 5ˊ exonuclease action that
removes mismatched nucleotide.
• This activity removes 99 out of every 100 mismatched bases.
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TERMINATOR RECOGNIZING
SEQUENCES
• At the last stage of termination, two replication fork meets at terminator
recognizing sequences, called as a Ter.
• Ter sequences with TUS protein create a complex which arrests the
replication fork.
• At this complex, the process of replication is completed and all other
proteins and enzymes leave this site.
• Only DNA topoisomerase II remains in action, it cuts both strands,
dissociates Ter-TUS complex and two different circular DNA is generated
44

RATE OF REPLICATION
The replication of an entire bacterial chromosome in
approximately 40 minutes at 37°C requires that each
replication fork move about 1000 nucleotides per second.
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REFERENCES
 Gerald Karp (2010). Cell and molecular biology:
concepts and experiments (6th ed.). John Wiley &
sons. ISBN-13 978-0-470-48337-4.
 https://www.mun.ca/biology/scarr/Meselson_StahL_
experiment.html
 https://geneticeducation.co.in/prokaryotic-dna-
replication/
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Dna replication prokaryotes

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