Roll no. : 1901BS085 ; Name: C.

Lalenpuii Zorem

PROTEINS INVOLVE IN DNA REPLICATION

DNA replication is a biological process in which a double-stranded DNA molecule is copied to produce two
identical molecules. DNA replication occurs in three steps: 1) Initiation 2)Elongation 3) Termination. The
mechanism of DNA replication is very similar in most organisms, however, differences exist with respect to
the enzymes and proteins involved. These differences can be seen in the DNA replication process in
prokaryotes and eukaryotes.

MAJOR PROTEINS/ENZYMES IN DNA REPLICATION

IN PROKARYOTES :

Proteins/enzymes involve in initiation phase:

 DnaA Protein: It is a replication initiation factor. It promotes the unwinding of DNA at oriC ( origin of
chromosomal DNA). The concentration of DnaA determines the onset of initiation phase of DNA replication.
DnaA proteins bind to the DnaA box (which is present within oriC) with the help of other DNA-binding
proteins like HU and IHF.

>HU: It is a small, basic, heat-stable DNA-binding protein, it serves as an accessory factor to assist the
binding of DnaA proteins to oriC, for the initiation of DNA replication.

>IHF: It is an Integration Host Factor, and can bend the DNA around the DnaA proteins, which result, in
the separation of the AT-rich region in the oriC.

 DnaB protein: It is a DNA helicase, that can break hydrogen bonds present between two DNA strands. It
travels along the DNA in a 5’ to 3’ direction. It initiates the replication of bacterial chromosome in both
directions (bidirectional replication). It obtains its energy by the hydrolysing ATP in order to separate the ds-
DNA.

 DnaC protein: It is a loading factor that mediates the loading of DnaB to DnaA.

 Single-stranded binding protein (SSBP): It temporarily stabilize the unwound state of DNA , by binding to
the parental DNA and prevent them from re-forming a double helix. SSBPs protect the ssDNA from nucleases
until it can be replicated, and it also prevents the formation of secondary structure in the single stranded DNA.
  DNA Gyrase:: It is an enzyme known as topoisomerase (type II),, which travels in front of DNA helicase and
removes positive supercoiling. It relieves strain while DNA is being unbinded by helicase.

Figure : Initiation of Replication of DNA and proteins/enzymes involve:

Proteins/enzymes involve in Elongation:

 DnaG protein : This gene produces RNA polymerase called primase. The RNA polymerase synthesizes new
RNA strands called oligonucleotides which are typically 10-60
60 nucleotides in length. The oligonucleotides
serve as primers,, or starting points, for DNA synt
synthesis
hesis by bacterial DNA polymerase III. DnaG serves an
important role because DNA polymerase cannot initiate the synthesis of a DNA strand on its own, but can only
add nucleotides to a pre-existing
existing strand. A single RNA primer is synthesized at the origin of replication, this
primer then serves to prime leading strand DNA synthesis. For the lagging strand DnaG synthesizes an RNA
primer every few kilobases, these primers then serve as substrates for the formation of Okazaki fragments.
 Primosome : Primosome is a protein complex formed by the helicase and primase, it synthesizes RNA primers
on both single stranded strands of DNA
DNA,, once on the leading strand, and repeatedly on the lagging strand
initiating each Okazaki fragment.. The primosome consists of seven proteins: 1.DnaG primase 2.DnaB helicase
3.DnaC helicase assistant 4.DnaT 5.PriA 6.PriB 7.PriC. The primary role of Primosome is to recruit the
replicative helicase onto single-stranded
stranded DNA.
 DNA polymerase III: It catalyzes the addition of deoxyribonucleotides to RNA primers either continuously
on the leading strand or discontinuously by the synthesis of Okazaki fragments on the lagging strand. As the
DNA polymerase reads the template strand, it selects complementary nucleotides for the new strand based on
hydrogen capability. It terminates an Okazaki fragment when it bumps into the RNA primer of the preceding
Okazaki fragments.

 DNA polymerase I: It joins the Okazaki fragments by removing the RNA primers and replacing with DNA, it
also supports repair of damaged DNA.

 RNaseH : It is an endogenous enzyme that cleavesthe RNA strand of an RNA

RNA-DNA
DNA duplex. It excises the
RNA primer along DNA polymerase I..

 DNA ligase: It facilitates the joining of DNA strands together by filling the gaps (nicks) with
deoxyribonucleotides.
 Figure: Elongation phase of Replication
lication of DNA and proteins/enzymes involve:

Proteins/enzymes involve in Termination phase :

 TUS (termination utilization substance): It is a protein that binds to the terminator sequence and stops the
movement of the replication forks. Tus acts as a counter
counter-helicase
helicase when it comes in contact with an advancing
helicase.In
In E.coli the termination of replication occu
occurs
rs at variable sites within regions. TerA, TerD and TerE
block the movement of replicating forks advancing in the counterclockwise and TerC, TerB, and TerF block the
clockwise movement.

FIGURE: Termination of replication of DNA and proteins/enzymes involve:

 IN EUKARYOTES:

Proteins/enzymes involve in initiation phase :

 ORC protein (Origin Enriched Sequence): ORC protein complex bind to the origin of replication. ORC
serves as the base for the assembly of the pre-replication complex (Cdc6, Tah11 also known as Cdt1 and the
Mcm2-Mcm7 complex). The ORC is present throughout the cell cycle, binding the origin of replication, but it
is active only in late mitosis and early G1 phase.
 Cell division cycle6 (Cdc6): It is the part of pre-replicative complex. The function of Cdc6 is to assemble pre-
replicative complex at the origin of replication. It also activates and maintain the checkpoint mechanisms that
coordinate S-phase and mitosis. Cdc6 loads Mcm to ORC.
 Chromosome Licensing and DNA Replication Factor 1(Cdt1): It is part of the pre-replication complex, it
binds to the ORC along with Cdc6 and Mcm.

 Mcm (Mini Chromosome Maintainance): Mcm2-Mcm7 proteins are capable of associating into a ring-
shaped complex that possesses helicase activity. Just before the beginning of S-phase of the cell cycle, the
activation of key protein kinases leads to the activation of the Mcm2-Mcm7 and the initiation of replication.

The enzyme helicase separates the parental strands and open the replication fork. The Mcm proteins move
with the replication fork and are essential for the completion of replication of a replicon.

 Replication Protein A: It is a heterotrimeric single-stranded DNA binding protein that binds to the exposed
single-stranded DNA template. RPA also interacts with single-stranded DNA with proteins to coordinate DNA
replication, repair and recombination.
 DNA topoisomerase II: It cuts the double helix of DNA, both the strands simultaneously in order to remove
the positive supercoils in DNA.
 Figure: Initiation phase of DNA replication and proteins/enzymes involve:

Proteins/enzymes involve in Elongation:

 Proliferating Cell Nuclear Antigen (PCNA

(PCNA): It is a DNA Clamp that acts as Processivity Factor for DNA
Polymerase Delta.
 DNA polymerases:
α (alpha): It initiates the DNA replication at the origin of replication, on both of the leading and lagging strands,
it helps in synthesis of Okazaki fragment in the 5’ to 3’ direction.
β (Beta): It catalyzes the DNA synthesis during base excision repair, this base excision repair helps in repairing
damaged DNA, and plays an important role in maintaining genome stability.
δ (Delta): It synthesizes the DNA on the lagging strand (discontinuous DNA synthesis), it also catalyzes the
specific replication of a majority of the genome. It also helps in the repairing of DNA.
ε (Epsilon): It helps in the synthesis of nucleotide excision and base eexcision repair.
 FIGURE: Elongation phase in DNA replication and proteins/enzymes involve:

Proteins/enzymes involve in Termination phase:

 Telomerase: It is an RNA-dependent DNA polymerase that synthesizes DNA using RNA as a template.
Telomerase add GC- rich sequence repeats to the 3’ end of DNA, and extends the overhanging of telomeric
DNA, as a result it reverses the loss of DNA from each round of replication.

FIGURE: Telomerase adding deoxyribonucleotides to the 3’ end of DNA strand.