ENZYMES AND PROTIENS INVOLVED IN
Dr. K. Jeevaratnam,
Reg no. 1236829,
Subject: Molecular Biology
Content Slide no
dnaA Protein 3
DNA Helicase 4
Single-strand- binding protein (SSBP) 5
DNA Polymerase III 7
DNA polymerase I 9
DNA ligase 10
Topoisomerase ІІ 11
Topoisomerase І 12
Differences between prokaryotic and eukaryotic enzymes 13
Recognizes ori C sequence.
Binds to 9mers sequences forming an Initiation complex.
Melts DNA strands at oriC 13mers in presence of ATP to
form open complex.
Opens up the duplex at the replication
fork to provide a single stranded template.
Helicase uses energy from the ATP to
break the hydrogen bonds holding the base
Shows processivity and polarity.
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.
Single-strand- binding protein (SSBP)
Single-strand- binding protein (SSB) binds to the single-stranded
portion of each DNA strand, inhibiting the strands from reannealing and
protecting them from degradation by nucleases.
E.Coli SSBP is a tetramer and cooperatively in sequence independent
In eukaryotic cells, a heterotrimeric protein called replication factor A
serves the role of SSB in DNA replication. 5
Primase synthesizes a short (about 10 nucleotides) RNA
primer in the 5‟ 3‟ direction to prime DNA chain elongation.
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”.
Korenberg coined the term primosome to refer to collection of
protiens needed to make primers. 6
DNA Polymerase III
DNA pol III Holoenzyme is
major replicative polymerase for
both leading and lagging strand
DNA polymerase III begins
synthesizing DNA in the 5‟
3‟direction, beginning at the 3‟
end of each RNA primer.
This is a multiprotein complex
that consists of 10 distinct
Hallmarks of pol III are its very
high catalytic potency and
Catalytic core of DNA pol III composed of α ,ε θ- subunits ,contains the
polymerase activity and a 3‟ 5‟ exonuclease for proofreading.
A dimer of β-subunit forms a ringshaped structure and act as sliding
The ϒ-complex is a group of five proteins that comprise clamp loader;
the clamp loader places the clamp on DNA.
Two core proteins are bound together by τ-subunit.
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.
Clamp and clamp loader
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.
Proteases such as subtilisin or trypsin cleave DNA pol І into two fragments
Klenowfragment and a smaller N-terminal fragment. 9
DNA ligase seals the "nicks"
by forming phosphodiester
bonds between the 3„OH and
5‟phosphate of adjacent
converting them to a
continuous strand of DNA.
The ligase enzyme from Ecoli
requires NAD as a cofactor,
where as that of Viruses and
Eukaryotes requires ATP.
Topoisomerase ІІMake a transient double-
strand break in the helix and
forms a covalent linkage.
DNA gyrase can introduce
negative supercoils into DNA
GyrA subunit cuts and rejoins
GyrB subunit provides
energy by ATP hydrolysis.
Inhibited by Nalidixic acid
and Novobiocin. 11
Produces a transient single strand break forming 5‟phospho
DNA pol 111 is
involved in invivo
replication of DNA.
DNA pol 1 is the
primer in Ecoli.
DNA pol α and δ are involved in
invivo replication of nuclear DNA
where as DNA pol gama for
DNA pol beta and Є functions as
DNA repair enzyme.
DNA pol α synthesizes primers for
both leading and lagging strands.
Differences between prokaryotic and
In Ecoli SSB
β subunit of DNA
pol 111 acts clamp
Gama subunit of
DNA pol 111 acts as
DNA helicase is
Eukaryotic topoisomerase 1
relaxes both positive and
RF-A is the eukaryotic SSB.
PCNA acts as clamp.
RF-C acts as clamp loading
DNA helicase A is associated
with DNA α , and DNA helicase
Є is associated with DNA pol Є
Text book of Molecular Biology By David Clark Genes VII
Text book of Lehninger Principles of Biochemistry, Fourth
Edition - David