Enzymes and proteins involved

in process of DNA replication

Enzymes and proteins involved in process of
DNA replication
• DNA Polymerases
• Helicases
• Gyrases / Topoisomerases
• Primases
• Single-Strand DNA-Binding Proteins
• DnaA Protein
• RPA (replication protein A) and
• RFC (replication factor C)
DNA Polymerases
• Arthur Kornberg discovered the first E. coli DNA polymerase in 1958.

• DNA polymerase I (pol I) is a single 102-kD polypeptide chain.

• It catalyzes three quite distinct reactions.

• It has a DNA polymerase activity, but it also has two different

exonuclease activities:

 3’→5’, exonuclease activity and

 5’→3’ exonuclease activity.

• The 3’→5’ activity is important in proofreading newly synthesized DNA

• The 5’→3’ exonuclease activity allows pol I to degrade a strand ahead of the
advancing polymerase also helps in primer removal and replacement

• Another important feature of pol I is that it can be cleaved by mild

proteolytic treatment into two polypeptides:

• a large fragment (the Klenow fragment), which has the polymerase and
proofreading (3’→5’ exonuclease) activities;

• and a small fragment with the 5’→3’ exonuclease activity.

Klenow fragment
• The Klenow fragment is frequently used in molecular biology when DNA
synthesis is required and destruction of one of the parental DNA strands,
or the primer, is undesirable.
• For example, the Klenow fragment is often used to perform DNA end-
filling and can also be used to sequence a DNA.
• On the other hand, the whole pol I is used to perform nick translation to
label a probe in vitro, because nick translation depends on 5’→3’
degradation of DNA ahead of the moving fork.
• Thomas Steitz and colleagues determined the crystal structure of the
Klenow fragment in 1987, giving the fine structure of a DNA-synthesizing
machine.

• The most obvious feature of the structure is a great cleft between two α-
helices.

• This is the presumed binding site for the DNA that is being replicated.

• In fact, all of the known polymerase structures, including that of T7 RNA

polymerase, are very similar, and have been likened to a hand.
• In the Klenow fragment, one a-helix is part of the “fingers” domain,
the other is part of the “thumb” domain, and the b-pleated sheet
between them is part of the “palm” domain.

• The palm domain contains three conserved aspartate residues that

are essential for catalysis.

• They are thought to coordinate magnesium ions that catalyze the

polymerase reaction.
Discovery of DNA pol II and III
• In 1969, Paula DeLucia and John Cairns isolated a mutant with a defect in
the polA gene, which encodes pol I.
• This mutant (polA1) lacked pol I activity, yet it was viable, strongly
suggesting that pol I was not really the DNA replicating enzyme.
• Instead, pol I seems to play a dominant role in repair of DNA damage.
• It fills in the gaps left when damaged DNA is removed.
• The finding that pol I is not essential spurred a renewed search for the
real DNA replicase, and in 1971, Thomas Kornberg and Malcolm Gefter
discovered two new polymerase activities:
• DNA polymerases II and III (pol II and pol III).
• Of the three DNA polymerases in E. coli cells, pol I, pol II, and pol III, only pol
III is required for DNA replication.
• Thus, polymerase III is the enzyme that replicates the bacterial DNA.
• In 1999 DNA polymerases IV and V were isolated.
• These have unusual type of DNA repair.
• In E. coli, five distinct proteins function as DNA polymerases and are
designated polymerase I, II, III, IV, and V.
• DNA polymerases I and III are involved in normal DNA replication, whereas
DNA polymerases II, IV, and V play a role in DNA repair and the replication
of damaged DNA.
DNA polymerase III
•  DNA polymerase III is much more complex than DNA polymerase I,
having ten types of subunits.
• Its polymerization and proofreading activities reside in its α and ε
(epsilon) subunits, respectively.
• The θ subunit associates with α and ε to form a core polymerase,
which can polymerize DNA but with limited processivity.
• Two core polymerases can be linked by another set of subunits, a
clamp-loading complex, or complex, consisting of five subunits of
four different types, .
• 
• The core polymerases are linked through the (tau) subunits.

• Two additional subunits, (chi) and (psi), are bound to the clamp-loading complex.

• The entire assembly of 13 protein subunits (nine different types) is called DNA
polymerase III*

• and (not shown in figure),also bind to the complex.

• Two clamps interact with the two-core subassembly, each clamp a dimer of the
subunit.

• The complex interacts with the DnaB helicase through the subunit.
• DNA polymerase III* can polymerize DNA, but with a much lower
processivity than one would expect for the organized replication of an
entire chromosome.
• The necessary increase in processivity is provided by the addition of the
subunits, four of which complete the DNA polymerase III holoenzyme.
• The subunits associate in pairs to form donut-shaped structures that
encircle the DNA and act like clamps.
• Each dimer associates with a core subassembly of polymerase III* (one
dimeric clamp per core subassembly) and slides along the DNA as
replication proceeds.
• The sliding clamp prevents the dissociation of DNA polymerase III from
DNA, dramatically increasing processivity—to greater than 500,000
Eukaryotic DNA polymerases
DNA polymerase α
• The
  replication of nuclear chromosomes involves DNA polymerase α, in
association with DNA polymerase.
• DNA polymerase α is typically a multi subunit enzyme with similar structure
and properties in all eukaryotic cells.
• One subunit has a primase activity, and the largest subunit (Mr ~180,000)
contains the polymerisation activity.
• However, this polymerase has no proofreading 3’5’ exonuclease activity,
making it unsuitable for high-fidelity DNA replication.
• DNA polymerase α is believed to function only in the synthesis of short primers
(containing either RNA or DNA) for Okazaki fragments on the lagging strand.
DNA polymerase 𝛿
• These
  primers are then extended by the multi subunit DNA polymerase .
• This enzyme is associated with and stimulated by a protein called proliferating
cell nuclear antigen (PCNA; Mr 29,000), found in large amounts in the nuclei of
proliferating cells.
• The three-dimensional structure of PCNA is remarkably similar to that of the β
subunit of E. coli DNA polymerase III
• PCNA has a function analogous to that of the β subunit, forming a circular
clamp that greatly enhances the processivity of the polymerase.
• DNA polymerase has a 3’5’ proofreading exonuclease activity and appears to
carry out both leading and lagging strand synthesis in a complex comparable to
the dimeric bacterial DNA polymerase III.
DNA polymerase 𝜀
• 
• DNA polymerase , replaces DNA polymerase in some situations, such
as in DNA repair.

• DNA polymerase may also function at the replication fork, perhaps

playing a role analogous to that of the bacterial DNA polymerase I,
removing the primers of Okazaki fragments on the lagging strand.
DNA Helicases

• The enzyme that harnesses the chemical energy of ATP to separate the
two parental DNA strands at the replicating fork is called a helicase.
• The helicase that unwinds double stranded DNA at the replicating fork is
encoded by the E. coli DnaB gene.
• The hexameric helicases have a characteristic ring-shaped structure
• Helicases use ATP energy to separate the two parental DNA strands at
the replicating fork.
• Many DNA helicases have been identified in E. coli cells.
• The first three to be investigated are the rep helicase, and DNA helicases
II and III
Dna-B proteins (Mw- 3,00,000)
• It is a Primosome constituent and consists of six subunits.
• It unwinds DNA during replication.
• It is responsible for the extension of open complex during replication.
Rep Proteins (Mw 65,000)
• It is a helicase consisting of one subunit.
• It binds to the 5’ 3' template and moves in 3’ 5' direction.
• It actively participates in leading strand synthesis in replication.
DNA Helicase - II (Mw 75,000)
• It is helicase consisting of only one subunit.
• It binds with 3’5' template stand and moves along in 5’ 3’ direction.
• It is involved in lagging strand synthesis in replication.
DNA GYRASE
• DNA gyrase, often referred to simply as gyrase, is an enzyme that
relieves strain while double-strand DNA is being unwound by helicase.
• This causes negative supercoiling of the DNA.
• The ability of gyrase to relax positive supercoils comes into play during
DNA replication.
• DNA gyrase belongs to a class of enzymes called topoisomerases that
introduce transient single- or double-stranded breaks into DNA and
thereby allow it to change its shape, or topology
• Type II topoisomerases, DNA gyrase of E. coli break and reseal both
DNA strands.
PRIMER
• A primer is a strand of nucleic acid that serves as a starting point for
DNA synthesis.
• It is required for DNA replication because the enzymes that catalyze
this process, DNA polymerases, can only add new nucleotides to an
existing strand of DNA.
• The polymerase starts replication at the 3'-end of the primer, and
copies the opposite strand.
• In most cases of natural DNA replication, the primer for DNA synthesis
and replication is a short strand of RNA.
Primases

• Primase is an enzyme that synthesizes short RNA sequences called primers.

• These primers serve as a starting point for DNA synthesis.

• Since primase produces RNA molecules, the enzyme is a type of RNA

polymerase.

• Primase functions by synthesizing short RNA sequences that are complementary

to a single-stranded piece of DNA, which serves as its template. [DNA dependent
RNA polymerase]
• It is critical that primers are synthesized by primase before DNA
replication can occur.
• This is because the enzymes that synthesize DNA, which are called
DNA polymerases, can only attach new DNA nucleotides to an existing
strand of nucleotides.
• Therefore, primase serves to prime and lay a foundation for DNA
synthesis.
• The crystal structure of primase in E. coli with a core containing the
DnaG protein was determined in the year 2000.
• The DnaG and primase complex is cashew shaped and contains three
subdomains.
PRIMOSOME
• The prepriming proteins, DNA helicase and primase are sequentially
assembled on the template DNA to generate a primosome.
• It is a complex containing Pri A, Pri B, Pri C, Dna T, Dna B, Dna C and
Primase (Dna G) proteins.
• Primososme complex without primase referred as preprimosome
complex.
• Primosome complex plays a vital role in lagging strand synthesis by
synthesizing the primer at frequent intervals.
• Of all the proteins actual primer synthesizing ability relies with primase
molecule.
• But each protein of the complex has
their own role i.e. each of them
necessary for the function of
primosome complex in replication
for example.
• Pri A, Pri B, Pri C necessary for the
binding of primosome complex to
DNA.
• Dna B responsible for helicase
activity.
• Dna C aids the binding of Dna B.
• Dna T helps the binding of DnaB and
Dna C complex to Pri A, Pri B and Pri
C complex.
Single-Strand DNA-Binding Proteins

• It was called a DNA-unwinding protein (M.W. 22,000).

• Single-stranded DNA-binding protein, or SSB, binds to single-stranded

regions of DNA to prevent premature annealing, to protect the single-
stranded DNA from being digested by nucleases, and to remove
secondary structure from the DNA to allow other enzymes to function
effectively upon it.
• Single-stranded DNA is produced during all aspects of DNA
metabolism: replication, recombination and repair.

• As well as stabilizing this single-stranded DNA, SSB proteins bind to and

modulate the function of numerous proteins involved in all of these
processes.

• SSB proteins have been identified in both viruses and organisms from
bacteria to humans.
• In E. coli it contains four subunits.
• The major function of SSB is to prevent recoiling of DNA strands after
it’s unwinding by helicases.
• Thus, SSB plays vital role in replication.
 DnaA Protein

• DnaA is a protein that activates initiation of DNA replication in bacteria.

• It is a replication initiation factor which promotes the unwinding of DNA
at oriC.
• The onset of the initiation phase of DNA replication is determined by
the concentration of DnaA.
• Dna-A protein (Mw 48,000)
• It binds to 4 of 9 bp sequence and unwinds a 3 of 13 bp sequence at Ori
C site and forms an open complex during initiation of replication.
• It is the first protein which binds to DNA to initiate DNA replication.
RPA (replication protein A) and
RFC (replication factor C)
•  Two other protein complexes also function in eukaryotic DNA
replication.
• RPA (replication protein A) is a eukaryotic single-stranded DNA–
binding protein, equivalent in function to the E. coli SSB protein.
• RFC (replication factor C) is a clamp loader for PCNA and facilitates the
assembly of active replication complexes.
• The subunits of the RFC complex have significant sequence similarity
to the subunits of the bacterial clamp loading () complex.
 Proteins Involved in E. coli DNA Replication
Protein Function
DnaA protein Recognizes ori sequence; Binds to DnaA boxes within the origin to initiate
DNA replication; opens duplex at specific sites in origin
DnaB protein (helicase) Separates double-stranded DNA(unwind DNA)
DnaC protein Aids DnaA in the recruitment of DNA helicase to the origin
HU Histone like protein; DNA-binding protein; stimulates initiation
Primase (DnaG protein) Synthesizes short RNA primers
Single-strand binding protein Binds to single-stranded DNA and prevents it from re-forming a double-
stranded structure
DNA gyrase (DNA topoisomerase Removes positive supercoiling ahead of the replication fork
II)
DNA polymerase III Synthesizes DNA in the leading and lagging strands
DNA polymerase I Removes RNA primers, fills in gaps with DNA
DNA ligase Covalently attaches adjacent Okazaki fragments
Tus Binds to ter sequences and prevents the advancement of the replication
fork
Dam methylase Methylates (5’)GATC sequences at oriC
The proteins involved with DNA replication.
Functions of key proteins involved with DNA
replication
• DNA helicase breaks the hydrogen bonds between the DNA strands.
• Topoisomerase alleviates positive supercoiling.
• Single-strand binding proteins keep the parental strands apart.
• Primase synthesizes an RNA primer.
• DNA polymerase III synthesizes a daughter strand of DNA.
• DNA polymerase I excises the RNA primers and fills in with DNA (not
shown).
• DNA ligase covalently links the Okazaki fragments together.
References
• Brooker, J. R. 2012. Genetics Analysis and Principle 4th Edition. Hill
International edition. McGraw.
• Fisher, M. (2001). Lehninger Principles of Biochemistry, ; By David L.
Nelson and Michael M. Cox. The Chemical Educator.
• Brown, T. A. (2007). Genomes 3. Garland Science Publishing
• Weaver, R. F. (2021). Molecular Biology, Fifth Edition. Hill International
edition. McGraw.