DNA REPLICATION IN

PROKARYOTES
Praveen Deepak
Assistant Professor of Zoology
S. S. College, Jehanabad

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Introduction
 The prokaryotic chromosome is a circular molecule.
 They are supercoiled structure that are suspended in cytoplasm.
 They are negatively supercoiled during normal growth.
 Most prokaryotes contain single circular genome, however some have multiple
copies of genome, such as Vibrio cholerae (Cholera) contains 2 genomes (Trucksis
et. al., 1998) and Borrelia burgdorferi (Lyme disease) contains up to 11 copies of
genome (Ferdows & Barbour, 1989).
 Borrelia burgdorferi genome are not supercoiled as that of Escherichia coli (E. coli);
rather; their DNA strands are diffused throughout the cell (Hinnebusch & Bendich,
1997).
 In addition to genome, most prokaryotes also contain linear or circular small DNA
molecules known as plasmid, an extrachromosomal DNA molecule that encode
nonessential genes. It may be found in one or multiple copies in the bacteria.
Borrelia may contain up to 20 plasmids (Fraser et al., 1997).
 Plasmids are much smaller than genome often contain only 1500 kb and replicate
independently.
 Replication, which is a duplication of chromosome, starts from a sequence known
as the origin of replication (point at which the DNA opens up) in prokaryotes.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Introduction
 The specific structure of the replication origin varies somewhat from
species to species, but all share some common characteristics such as
high AT content.
 Prokaryotes have single replication origin per circular chromosome except
Archaea have several replication origin. It is called as OriC in E. coli.
 It is called as theta replication as the structure resembles the Greek
alphabet theta (θ).
 The replication in prokaryotes occurs in three
steps:
1.) Initiation: at replication origin
2.) Elongation: at replication fork
3.) Termination: at termination sequence
 Replication is bidirectional Y-shaped formation 5'-to-3'
of replication fork runs in both directions. direction
 Replication is semi-continuous:
Leading strand – continuous
Lagging strand – fragmented (Okazaki fragments)

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Mode of replication
 Replication is performed by semiconservative mode, i.e., one strand of DNA is
conserved (template strand) while other strand is not conserved.

Meselson and Stahl,

All cells

Donnianni and
Symington, and
Saini et al.

No evidence of its
occurrence in nature

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Initiation
 DNA replication starts with the formation of replication origin which is highly conserved
among bacteria.
 The oriC replication origin of E. coli 245 base pairs.
 The key sequences oriC are two series of short repeats; three repeats of a 13 base pair
sequence and four repeats of a 9 base pair sequence.

 DnaA protein binds to 9-mer DnaA box consensus sequence, 5′-TTATnCACA-3′ and
results in unwinding of DNA at oriC. (DiaA complex : DiaA-DnaA complex)
 Unwinding results in the recognition of DNA by other replication proteins that act
subsequently in the initiation process.
 Thereafter, integration host factor binds to its single site, which causes a sharp (120–180°)
bend in the double helix (Swinger and Rice, 2004).
 A complex consisting of oriC, IHF, DiaA, and oligomeric ATP-DnaA is considered to make
up the initiation complex in E. coli (Keyamura et al., 2007, 2009).
 This complex finally leads to the loading of DNA helicases and thereby the formation of
replisomes.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Initiation
Unbinding of DNA by helicases

An enzyme that unwinds the double

helix by breaking the hydrogen bonds
between the complimentary bases

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Initiation
 Replisome – DNA ploymerases, helicases, SSBs, DNA
ligase, clamps (e.g. topoisomerases).
 Replisome is a multienzyme complex of >1MDa.

http://www.bioinfo.org.cn/book/biochemi
stry/chapt24/bio3.htm

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Initiation
Regulation of initiation of replication

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 Elongation of new DNA at a replication fork.
 It is catalyzed by enzymes called DNA polymerases, which add nucleotides to the
3' end of a growing strand.

DNA polymerase adds

nucleotides to the deoxyribose
(3') ended strand in a 5' to 3'
direction

Accuracy
1 error in 1 billion bases

Speed
500 nt/s in bacteria
50 nt/s in mammals

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 DNA is synthesized in 5´to 3´direction

Bacterial chromosome
doubles in 40 min

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
RNA polymerase or DNA primase

 Makes the 10 nt RNA primer

required for start of replication
in the beginning of leading
strand.

 In beginning of each Okazaki-

Fragment, it synthesizes primer
sequences of ribose nucleotide.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 Topoisomerase in initiation complex prevents over-winding of the DNA double helix
ahead of the replication fork as the DNA is opening up by breaking the strand.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 Nucleotides are added by the DNA polymerase.

DNA polymerase requires:

1. A free 3’-OH group

supplied by RNA Primer
for start of
polymerisation

2. Mg2+ ions for activity in

active site

3. A template to copy

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 Nucleotides are added by the DNA polymerase. DNA polymerase is a protein
complex like DNA polymerase III composed of 10 subunits as shown below.

Structure of DNA polymerase III (dnaX)

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
Binding of SSBs proteins to DNA

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
Binding of SSBs proteins to DNA

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
E. coli contains multiple DNA polymerases

DNA poly I DNA poly II DNA poly III

Number/cell 400 100 10
Speed (nt/s) 16-20 2-5 250-1000
3' exonuclease Yes Yes No
5' exnuclease Yes No No
Processitivity 3-200 10000 500000
Role DNA repair DNA repair Replication
RNA primer removal
 DNA poly I was found by Arthur Kornberg at mid 1950’s
 It has three enzymatic activities:
• Polymerase activity
• 3’ to 5’ exonuclease activity
• 5’ to 3’ exonuclease activity
Klenow enzyme is lacking one subunit responsible for the 5’ to 3’ exonuclease activity.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 DNA is synthesized in the replication fork in 5’ to 3’ direction

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 DNA is synthesized in the replication fork in 5’ to 3’ direction

 Here, a primer sequence is added

with complementary RNA
nucleotides by RNA polymerase
called primase, which are then
replaced by DNA nucleotides.
 DNA polymerase adds DNA
nucleotides to the 3′ end of the
newly synthesized polynucleotide
strand.
 The template strand specifies which
of the four DNA nucleotides (A, T, C,
or G) is added at each position along
the new chain.
 Only the nucleotide complementary
to the template nucleotide at that
position is added to the new strand.
 Once DNA replication is finished, the
daughter molecules are made
entirely of continuous DNA
nucleotides, with no RNA portions.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 Leading strand synthesis is continuous whereas lagging strand is synthesized in
fragments

 DNA polymerase can only synthesize new strands in the 5′ to 3′ direction.

 The “leading strand” is synthesized continuously toward the replication fork as helicase
unwinds the template double-stranded DNA.
 The “lagging strand” is synthesized in the direction away from the replication fork and
away from the DNA helicase unwinds.
 This lagging strand is synthesized in pieces because the DNA polymerase can only
synthesize in the 5′ to 3′ direction. The pieces are called as Okazaki fragments
 Length of Okazaki fragments in prokaryotes are 1000-2000 nt.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
 Error correction or proofreading: Any error that has been left during the
replication is corrected by DNA polymerase.
 Error repair is achieved with the exonuclease activity of DNA polymerase.

This results in a very low error rate of 1 in 1 billion nucleotides

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
Error correction
 3’ to 5’ exonuclease activity corrects errors

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
Sealing the nick of Okazaki fragments

 DNA ligase seals the nicks between

Okazaki fragments

 It requires close and free 3’-OH and 5’-P

and proper base-pairing

 NAD+ required in prokaryotes

ATP required in eukaryotes

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Elongation
Sealing the nick of Okazaki fragments – Nick sealing by DNA ligase

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Termination
Termination of replication takes place at the termination site in the prokaryotic
DNA
 DNA replication terminates
when replication forks reach
specific ‘termination sites’,
i.e. replication forks meet
each other on the opposite
end of the parental circular
DNA.
 The two replication forks are
synchronized by 10-23 bp Ter
sequences that bind Tus
proteins
 Tus proteins can only be
displaced by replisomes
Terminus Utilization Substance (Tus) coming from one direction

 Tus protein binds to terminator sequences (Ter sequence) and acts as a

counter-helicase when it comes in contact with an advancing helicase. The
bound Tus protein effectively halts DNA polymerase movement.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Enzymes and Proteins of Prokaryotic DNA Replication
Enzyme/protein Specific function
DNA pol I Exonuclease activity removes RNA primer and replaces
with newly synthesized DNA
DNA pol II Repair function
DNA pol III Main enzyme that adds nucleotides in the 5'-3' direction
Helicase Opens the DNA helix by breaking hydrogen bonds
between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments to create
one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Sliding Clamp Helps to hold the DNA polymerase in place when
nucleotides are being added
Topoisomerase Helps relieve the stress on DNA when unwinding by
causing breaks and then resealing the DNA
Single-strand binding Binds to single-stranded DNA to avoid DNA rewinding
proteins (SSB) back.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Steps of the DNA replication in prokaryotes
 DNA unwinds at the origin of replication.
 Helicase opens up the DNA-forming replication forks; these are extended
bidirectionally.
 Single-strand binding proteins coat the DNA around the replication fork to
prevent rewinding of the DNA.
 Topoisomerase binds at the region ahead of the replication fork to prevent
supercoiling.
 Primase synthesizes RNA primers complementary to the DNA strand.
 DNA polymerase starts adding nucleotides to the 3'-OH end of the primer.
 Elongation of both the lagging and the leading strand continues.
 RNA primers are removed by exonuclease activity.
 Gaps are filled by DNA pol by adding dNTPs.
 The gap between the two DNA fragments is sealed by DNA ligase, which
helps in the formation of phosphodiester bonds.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad

Steps of the DNA replication in prokaryotes

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Edition, McGraw Hill Publication, New York, USA

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of Bacteria, 2nd ed., ASM Press, Washington DC, USA, 2003.

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Microbial Physiology 24(5-6).

 Griswold A. 2008. Genome Packaging in Prokaryotes: the Circular Chromosome

of E. coli. Nature Education 1(1):57.

 Kuzminov A. 2014. The precarious prokaryotic chromosome. Journal of

Bacteriology 196(10):1793-1806.

Praveen Deepak, Assistant Professor of Zoology, Swami Sahjanand College, Jehanabad