3. PROTIENS INVOLVED IN
DNA REPLICATION:
3
• DNA POlYMERASE 𝜶: Addition of primer
• DNA POlYMERASE 𝝳 : Synthesis in lagging strand
• DNA POlYMERASE 𝟄 : Synthesis in leading strand
• SSBP : Single stranded binding protein
• RFC : Replication factor C : clamp loader
• ORC : Origin recognition complex
• MCM : Minichromosome maintainance complex
• CDK : cyclin dependent kinase
4. EUKARYOTES
4
• Initiation is the start process of replication.
• G1 phase is the preparation phase where, making of all proteins for replication are
present.
• In S phase the actual DNA replication takes place.
• The replication process in DNA occurs by the separation of two strands by certain
enzymes in which each strand serves as a template in further processes.
• This type of process is referred to as the semiconservative replication process
because of the DNA strands after separation act as a template for new DNA
molecules for further synthesis.
• Before initiation can occur, the supercoiled chromosome must be relaxed.
• The alteration to the structure of the chromosome is accomplished by the enzyme
TOPOISOMERASE.
5. EUKARYOTES
5
• Signal from G1 phase to S phase is transmitted by CDK and CDt depending on their
concentration.
• The role of CDK is formation of pre existing initiation complex and activation of
existing initiation complex.
• However, how CDKs promote DNA replication has been a long-standing
question, because all the essential CDK substrates in DNA replication have
not been identified yet1.
• Recently, Sld2 and Sld3 were identified as essential substrates of CDKs in the
initiation step of DNA replication in budding yeast.
HOW DOES A CELL KNOW WHEN TO
INITIATE THE REPLICATION PROCESS?
6. EUKARYOTES
6
• The initial step of DNA replication of eukaryotic cells is to find a specific
chromosomal location called an origin of replication.
• Eukaryotes like in human, there consists of multiple sites of origin replication
therefore, the replication initiation protein identifies and binds to the specific site to
the nucleosomes. But in some eukaryotes like in yeast, they have a defined site of
replication.
• DNA synthesis always initiates at origins of replication –specific loci in the DNA
genome with high concentrations of A and T base pairs.
• This is because these base pairs have only 2 hydrogen bond links, while C and G
pairs have 3. Therefore, it takes less energy to separate the DNA strands at these
locations.
INITIATION PROCESS:
7. EUKARYOTES
7
• As cells enter the G1 phase of the cell cycle, the Origin
recognition complex or ORC bound to ATP comes and binds
to the origin replication site.
• This ORC recruits ATP-bound helicase loading protein CDC6.
• Two copies of hexamer Mini-chromosome maintenance
Protein complex or MCM 2-7 complex attach alongside
another helicase loading protein, CDT1.
• This makes the whole complex unstable and this triggers
ATP hydrolysis by cdc6.
• These results in the MCM complex encircling the double-
stranded DNA and the release of cdc6 and cdt1 from the
origin.
• Here MCM complex functions as helicase (unwinds the
DNA)
8. EUKARYOTES
8
• As the cell enters the S phase of the cell cycle initiation
of replication starts.
• This starts with the activation of two protein kinases,
cyclin-dependent kinase or CDK and Dbf4-dependent
kinase or DDK.
• Once activated, DDK phosphorylates the mcm2-7
complex. Whereas, CDK phosphorylates two other
replication proteins sld2 and sld3.
• Phosphorylated sld2 and sld3 then bind to Dpb11 and
they all together attach to the origin complex.
• This facilitates the binding of helicase-activating
proteins, CDC 45 and GINS to the complex.
• cdc45 and GINS form a stable complex with the MCM2-
7 helicase which is called as CMG complex.
• This complex activates MCM2-7 helicase activity.
• Before unwinding DNA polymerase is recruited to the
helicase at both strands. Where DNA polymerase δ in
lagging strand and ε leading strand
• After that, the DNA elongation begins.
9. EUKARYOTES
9
• Replication on each strand of DNA involves
DNA polymerase and a sliding clamp PCNA
(proliferating cell nuclear antigen)
• PCNA forms a ring around the DNA to
which the DNA polymerase delta binds.
• Formation of this ring requires another
factor i.e. RFC (Replication factor C)
• RFC recognizes the interaction and region
between primer and nucleotide added by
the α polymerase.
• The α polymerase adds the first 20-25
nucleotides after the RNA primer.
• Without PCNA ring long stretch of
nucleotides cannot be achieved.
10. REFERANCES :
• Steps of DNA Replication in Eukaryotes - The Science Notes
• Molecular Biology of the Gene, 6th Edition (2008), James D. Watson, Tania Baker, Stephen P.
Bell, Alexander Gann, Michael Levine, Richard Lodwick. Pearson Education, Inc. and Dorling
Kindersley Publishing, Inc. USA
• Tanaka, S., Araki, H. (2016). Role of CDK in Replication Initiation. In: Kaplan, D. (eds) The
Initiation of DNA Replication in Eukaryotes. Springer, Cham. https://doi.org/10.1007/978-3-
319-24696-3_13
• Reußwig, KU., Boos, D., Pfander, B. (2016). Roles of Sld2, Sld3, and Dpb11 in Replication
Initiation. In: Kaplan, D. (eds) The Initiation of DNA Replication in Eukaryotes. Springer,
Cham. https://doi.org/10.1007/978-3-319-24696-3_15
• Masumoto, H., Muramatsu, S., Kamimura, Y. et al. S-Cdk-dependent phosphorylation of Sld2
essential for chromosomal DNA replication in budding yeast. Nature 415, 651–655 (2002).
https://doi.org/10.1038/nature713
• https://youtu.be/ZsayFrGuf-I?si=njZQmteOQlxJGymX
• https://youtu.be/8tYgCbDV3j4?si=HrTcas5SPAthJdmv