Enzymes and proteins in DNA
replication
Presented by
R.Parthasarathy

Introduction
• Multiple proteins are required for DNA
replication at a replication fork.
• These include DNA polymerases, single-strand
DNA binding proteins, helicases,
primase,topoisomerases, and DNA ligase.
Some of these are multisubunit protein
complexes.

dnaA Protein
• The base sequence at the origin of replication
is recognized and bound by the dna A protein.

Helicase
• Helicase uses energy from the ATP to break the
hydrogen bonds holding the base pairs together.
• This allows the two parental strands of DNA to
begin unwinding and forms two replication
forks.
• Each strand of parental DNA has it own helicase.
• In humans, two inherited diseases, Werner's
syndrome and Bloom's syndrome, result from
helicase defects.
• E. coli contains at least 6 different helicases--
some involved in DNA repair and others in
conjugation, the principal helicase in DNA
replication is DnaB

SSB Protein
• Single-stranded DNA binding protein (SSB) binds to the
single-stranded portion of each DNA strand, preventing the
strands from reassociating and protecting them from
degradation by nucleases.
• gp32, the most studied SSB protein, binds in a strongly
cooperative fashion to single-strand DNA.
• That is, binding adjacent to another gp32 is much more likely
than the binding of a single gp32 in isolation.
• This property helps promote the denaturation of duplex DNA and
helps keep the DNA template in an extended, single-strand
conformation, with the purine and pyrimidine bases exposed so
that they can base-pair readily with incoming nucleotides.
• In E. coli, the protein is called ssb.
• In eukaryotic cells, a heterotrimeric protein called replication
factor A serves the role of SSB in DNA replication.

Primase
• Primase is an enzyme that copies a DNA template
strand by making an RNA strand complementary to it.
• Primase synthesizes a short (about 10 nucleotides)
RNA primer in the 5’ 3’ direction.
• The parental strand is used as a template for this
process.
• RNA primers are required because DNA polymerases
are unable to initiate synthesis of DNA, but can only
extend a strand from the 3' end of a preformed
“primer”
• The enzyme is active only in the presence of other
proteins (including a helicase), which create a complex
called the primosome

DNA polymerase III
• It catalyzes the chemical reactions for
polymerization of nucleotides.
• DNA polymerase III begins synthesizing DNA
in the 5’ 3’direction, beginning at the 3’
end of each RNA primer.
• The newly synthesized strand is
complementary and antiparallel to the
parental strand used as a template.

DNA polymerase I
• DNA polymerase I and RNAse H are involved in removing RNA
primers in the processing of DNA after replication.
• This enzyme removes the ribonucleotides one at a time
from the 5' end of the primer (5‘ 3' exonuclease).
• DNA polymerase I also fills in the resulting gaps by
synthesizing DNA, beginning at the 3' end of the
neighbouring Okazaki fragment.
• Both DNA polymerase I and III have the ability to
"proofread" their work by means of a 3' 5' exonuclease
activity.
• If DNA polymerase makes a mistake during DNA synthesis,
the resulting unpaired base at the 3' end of the growing
strand is removed before synthesis continues.

Comparison of DNA and RNA
polymerases
DNA Polymerase RNA Polymerase
Nucleic acid synthesized DNA RNA
(5’  3’)
Required template DNA* DNA*
Required substrates dATP, dGTP, dCTP, dTTP ATP, GTP, CTP, UTP
Required primer RNA (or DNA) None
Proofreading activity Yes No
(3’  5’ exonuclease)

Clamps and clamp loaders
• Protein from the DNA polymerase III
holoenzyme
complex holds the polymerase to the DNA.
• This helps the DNA polymerase complex to
stay on the DNA through an entire cycle of
replication.
• A multi subunit entity called the complex
functions as the "clamp loader". That is, it
loads the clamp onto the DNA.

Clamps and clamp loaders
• a protein dimer that encircles the DNA strand
and helps hold the DNA polymerase to the
DNA strand.
• In eukaryotic cells, a multi-subunit protein
called replication factor C (RF-C) is the clamp
loader, and proliferating cell nuclear antigen
(PCNA) is the sliding clamp.

DNA ligase
• DNA ligase seals the "nicks" between
Okazaki fragments, converting them to a
continuous strand of DNA.
• Covalently closes nicks in double-stranded
DNA.

DNA gyrase
• DNA gyrase (DNA topoisomerase II) provides a
"swivel" in front of each replication fork.
• As helicase unwinds the DNA at the replication
forks, the DNA ahead of it becomes overwound and
positive supercoils form.
• DNA gyrase inserts negative supercoils by nicking
both strands of DNA, passing the DNA strands
through the nick, and then resealing both strands
again.
• DNA topoisomerase I can relieve supercoiling in DNA
molecules by the transient breaking and resealing of
just one of the strands of DNA.

Action of a gyrase

Action of a type I topoisomerase

Step in Replication Prokaryotic cells Eukaryotic cells
Recognition of origin of Dna A protein Unknown
replication
Unwinding of DNA double helix Helicase Helicase
(requires ATP) (requires ATP)
Stabilization of unwound Single-stranded DNA-binding Single-stranded DNA-binding
template strands protein (SSB) protein (SSB)
Synthesis of RNA primers Primase Primase
Synthesis of DNA
Leading strand DNA polymerase III DNA polymerase δ
Lagging strand DNA polymerase III DNA polymerase α
Removal of RNA primers DNA polymerase I Unknown
(5 3' exonuclease)
Replacement of RNA with DNA DNA polymerase I Unknown
Joining of Okazaki fragments DNA ligase DNA ligase
(requires NAD) (requires ATP)
Removal of positive supercoils DNA topoisomerase II DNA topoisomerase II
ahead of advancing (DNA gyrase)
replication forks

References
MOLECULAR BIOLOGY by David Clark
Genes VII