DNA replication uses a semi-conservative method that results in two double-stranded DNA molecules, each with one old parental strand and one new daughter strand. Replication occurs through the theta and rolling circle mechanisms in prokaryotes. The theta mechanism involves unwinding DNA at the origin of replication and creating replication forks that allow bidirectional synthesis of new strands. The rolling circle mechanism involves nicking one strand at the origin, allowing it to be replicated unidirectionally as it "rolls" off the parental strand.

1. ACHARYA BANGALORE B-
SCHOOL
DEPT OF LIFE SCIENCE
Mahendra P
Bsc 4th Sem

2. DNA REPLICATION IN
PROKARYOTES

3. DNA REPLICATION
 DNA replication uses a semi-conservative method that results in a double-stranded DNA with one
parental strand and a new daughter strand.
 Watson and Crick’s discovery that DNA was a two-stranded double helix provided a hint as to how
DNA is replicated.
 During cell division, each DNA molecule has to be perfectly copied to ensure identical DNA
molecules to move to each of the two daughter cells.
 The double-stranded structure of DNA suggested that the two strands might separate during
replication with each strand serving as a template from which the new complementary strand for
each is copied, generating two double-stranded molecules from one.

5. Theta mode of replication
• DNA unwinds at the ori site from where the replication begins.
• It then creates the structure where the whole replicational machinery assembles.
• Since the structure resembles the Greek letter theta (θ), its name has been derived from it.
• The process gets initiated by the RNA primer.
• Then deoxyribonucleotides are added which extends the process.
• The replication process may proceed in one (unidirectional) or both directions (bi-directional).
• In the first case (unidirectional), a single replication fork moves around the circle until it returns to its point of origin. and then the two daughter DNAs
separate.
• In the other case (bidirectional replicational) two replication forks begin at ori then it travels to the opposite until they meet at some point on the other
side of the molecule.
• This is the most common mode of DNA replication.
• The theta mechanism is the most common form especially in Gram-negative bacteria like the proteobacteria.
• Commonly used plasmids, including ColE1, RK2, and F, as well as the bacteriophage P1, use this type of replication

8. Rolling circle mode of reolication
• It is called rolling-circle (RC) replication because it was first discovered in a type of phage where the template circle seems to “roll”.
• It is a unidirectional process (one direction only).
• Plasmids that replicate by this mechanism are sometimes called RC plasmids.
• This type of plasmid is found in the largest groups of bacteria, as well as in archaea.
• To perform this rolling-circle mode of replication, genetic material needs to be circular.
• In this method, one strand comes out while the other strand is being synthesized.
• Replication starts at the ori site that is the origin of replication where the Rep protein attaches one of the strands.
• Rep protein is the dimer that is formed of the two monomers.
• It has the tyrosine as the active group.
• First, the Rep protein recognizes and binds to the strand that contains the double-strand origin (DSO) on the DNA.
• Then the Rep protein can make a nick in the sequence.

9.  When the Rep protein has made a break in the DSO two ends will be formed in the DNA.
• At the 3’ end, there is the presence of OH group while at the 5’ end there is the presence of phosphate group.
• Rep protein will remain attached to the phosphate at the 5’ end of the DNA.
• Then the DNA polymerase III which is the replicative polymerase uses the free 3′ hydroxyl end at the break as a primer
to replicate around the circle.
• For the separation of the strand, it may use a host helicase.
• The Rep protein itself may have the helicase activity, depending on the plasmid.
• Once the circle is complete, the 5′ phosphate is transferred from the tyrosine on the Rep protein to the 3′ hydroxyl on the
other end of the strand. Then a single-stranded circular DNA is produced.

10. • This process is called a phosphotransferase reaction and requires little energy. The same reaction is used to re-form
a circular plasmid after conjugational transfer.
• The displaced circular single-stranded DNA now replicates by a completely different mechanism using only host-
encoded proteins.
• The RNA polymerase of the host cell recognizes the SSO ( single-strand origin) on the DNA.
• Then the RNA polymerase makes a primer.
• Then, replication occurs around the circle by DNA polymerase III.
• The RNA polymerase does not make this primer until the single-stranded DNA is completely displaced during the first
stage of replication.
• When the entire complementary strand has been synthesized, the DNA polymerase I remove the RNA primer with its
5′ exonuclease activity while simultaneously replacing it with DNA.
• DNA ligase joins the ends to make another double-stranded plasmid.
• Finally, The two new double-stranded plasmids are synthesized from the original double-stranded plasmid.

12. Bidirectional method of DNA replication
 In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of
the hydrogen bonds between the complementary strands, and synthesis of two new strands
by complementary base pairing.
 Replication begins at a specific site in the DNA called the origin of replication (oriC).
DNA replication is bidirectional from the origin of replication.
 To begin DNA replication, unwinding enzymes called DNA helicases cause short
segments of the two parent DNA strands to unwind and separate from one another at the
origin of replication to form two "Y"-shaped replication forks.
 These replication forks are the actual site of DNA copying

14.  All the proteins involved in DNA replication aggregate at the replication forks to form a replication complex
called a replisome.
 Single-strand binding proteins bind to the single-stranded regions so the two strands do not rejoin. Unwinding
of the double-stranded helix generates positive supercoils ahead of the replication fork.
 Enzymes called topoisomerases counteract this by producing breaks in the DNA and then rejoin them to form
negative supercoils in order to relieve this stress in the helical molecule during replication.
 As the strands continue to unwind and separate in both directions around the entire DNA molecule, new
complementary strands are produced by the hydrogen bonding of free DNA nucleotides with those on each
parent strand.
 As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA
polymerases join the nucleotides by way of phosphodiester bonds.
 Actually, the nucleotides lining up by complementary base pairing are deoxynucleotide triphosphates,
composed of a nitrogenous base, deoxyribose, and three phosphates. As the phosphodiester bond forms
between the 5' phosphate group of the new nucleotide and the 3' OH of the last nucleotide in the DNA strand,
two of the phosphates are removed providing energy for bonding.
 n the end, each parent strand serves as a template to synthesize a complementary copy of itself, resulting in the
formation of two identical DNA molecules

16.  In reality, DNA replication is more complicated than this because of the nature of the DNA polmerases.
 DNA polymerase enzymes are only able to join the phosphate group at the 5' carbon of a new nucleotide to the
hydroxyl (OH) group of the 3' carbon of a nucleotide already in the chain.
 As a result, DNA can only be synthesized in a 5' to 3' direction while copying a parent strand running in a 3' to 5'
direction.
 Each DNA strand has two ends. The 5' end of the DNA is the one with the terminal phosphate group on the 5'
carbon of the deoxyribose; the 3' end is the one with a terminal hydroxyl (OH) group on the deoxyribose of the 3'
carbon of the deoxyribose.
 The two strands are antiparallel, that is they run in opposite directions. Therefore, one parent strand - the one
running 3' to 5' and called the leading strand - can be copied directly down its entire length

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