The document details eukaryotic DNA replication, emphasizing its semi-conservative nature and complexity compared to prokaryotes. It outlines the processes involved, including DNA polymerase roles, various phases of the cell cycle, and the mechanisms of initiation, elongation, and termination of replication. Key proteins and enzymes, along with regulatory checkpoints and implications for cellular division and cancer, are also discussed.

1. eukaryoticDNAReplication
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. DNA replication is semi-conservative, one strand serves as the
template for the second strand. Furthermore, DNA replication
only occurs at a specific step in the cell cycle.
DNA replication in eukaryotes is much more complicated than
in prokaryotes, although there are many similar aspects.
DNA replication is a biological process that occurs in all living
organisms and copies their DNA; it is the basis for biological
inheritance.
Eukaryotic cells can only initiate DNA replication at a specific
point in the cell cycle, the beginning of S phase.
Due to the size of chromosomes in eukaryotes, eukaryotic
chromosomes contain multiple origins of replication
Introduction

3. History
Oswald Avery: Proved that DNA was the agent of transfer.
 Erwin Chargaff: determined that the bases A-T and C-G are
found in definite ratios.
Linus Pauling, Maurice Wilkins, and Roseland Franklin:
worked on the structure of DNA.
 Watson and Crick: determined the shape and structure of
DNA.

4. Definition
‘The process whereby a copy of a DNA molecule is made and
thus the genetic information it contains is duplicated.
The parental double stranded DNA molecule is replicated semi
conservatively, i.e. Each copy contains one of
the original strands paired with a newly synthesized strand that
is complementary in terms of AT and GC base pairing.’

5. DNA Polymerase
A DNA polymerase is a cellular or
viral polymerase enzyme that synthesizes DNA molecules from
their nucleotide building blocks.
 DNA polymerases are essential for DNA replication, and
usually function in pairs while copying one double-stranded
DNA molecule into two double-stranded DNAs in a process
termed semi conservative DNA replication.
DNA polymerases also play key roles in other processes within
cells, including DNA repair, genetic recombination, reverse
transcription, and the generation of antibody diversity via the
specialized DNA polymerase, terminal deoxynucleotidyl
transferase.

6. Classification
DNA polymerases are subdivided into seven different
families: A, B, C, D, X, Y, and RT.
1 Family A polymerases family include the extensively-
studied T7 DNA polymerase.
2 Family B Pol ζ (zeta) (polymerase ζ is a B Family
polymerase
3 Family C Polymerases are the primary bacterial chromosomal
replicative enzymes.
4 Family D Polymerases are still not very well
characterized.
5 Family X Contains the well-known eukaryotic
polymerase pol β, as well as other eukaryotic polymerases
such as pol σ, pol λ, pol μ &TdT

7. 6. Family Y Y Polymerases differ from others in having a
low fidelity on undamaged templates and in their ability
to replicate through damaged DNA.
7. Family RT (reverse transcriptase) The reverse
transcriptase family contains examples from both
retroviruses and eukaryotic polymerases.

8. Mechanism
The basic mechanisms of DNA replication are quite similar in
eukaryotes and prokaryotes.
 DNA replication is semi conservative and is continuous on one
strand and discontinuous on the other.
DNA replication in eukaryotes occurs only in the S phase of
the cell cycle.
Preparation in G1 phase
The first step in DNA replication is the formation of the pre-
initiation replication complex. The formation of this complex
occurs in two stages.

9. The first stage requires that there is no CDK activity. This can
only occur in early G1. The formation of the pre-RC is known as
licensing, but a licensed pre-RC cannot initiate replication in the
G1 phase.
Synthesis in S phase
Activation of the complex occurs in S-phase and requires Cdk2-
Cyclin E. Geminin binds to Cdt1 and sequesters it.
It is a periodic protein that first appears in S-phase and is
degraded in late M-phase, possibly through the action of
the anaphase promoting complex (APC).

10. Process of DNA Replication
DNA Replication proceeds in three enzymatically catalyzed
and coordinated steps: initiation, elongation and termination.
Origins
 This process is initiated at particular points in the DNA,
known as "origins", which are targeted by proteins that separate
the two strands and initiate DNA synthesis
 Origins contain DNA sequences recognized by
replication initiator proteins (e.g. the Origin Recognition
Complex in yeast). These initiators recruit other proteins to
separate the strands and initiate replication forks.

11. Four distinct mechanisms for synthesis have been described.
I. Many DNA viruses, phages and plasmids use a primase to
synthesize a short RNA primer with a free 3′ OH group
which is subsequently elongated by a DNA polymerase.
II. The retro elements (including retroviruses) employ a
transfer RNA that primes DNA replication by providing a
free 3′ OH that is used for elongation by the reverse
transcriptase.
III. In the adenoviruses the 3' OH group is provided by the side
chain of an amino acid of the genome attached protein to
which nucleotides are added by the DNA polymerase to
form a new strand.
IV. In the single stranded DNA viruses the many phages
and plasmids that use the rolling circle replication (RCR)
mechanism,

12. Proteins in DNA replication
DNA Helicase
Also known as helix destabilizing enzyme. Unwinds the DNA
double helix at the Replication Fork.
DNA Polymerase
Builds a new duplex DNA strand by adding nucleotides in the
5' to 3' direction. Also performs error correction.
Single-Strand Binding (SSB) Proteins
Bind to ssDNA and prevent the DNA double helix from re-
annealing after DNA helicase unwinds it thus maintaining the
strand separation.

13. DNA Ligase
Re-anneals the semi-conservative strands and
joins Okazaki Fragments of the lagging strand.
Primase
Provides a starting point of RNA (or DNA) for DNA
polymerase to begin synthesis of the new DNA
strand.
Telomerase.
Lengthens telomeric DNA by adding repetitive
nucleotide sequences to the ends of eukaryotic
chromosomes.

14. Replication fork
The replication fork is a structure that forms within the nucleus
during DNA replication. It is created by helicases, which break the
hydrogen bonds holding the two DNA strands together.
Leading strand
The leading strand is the template strand of the DNA double helix
so that the replication fork moves along it in the 3' to 5' direction.
On the leading strand, a polymerase "reads" the DNA and
adds nucleotides to it continuously. This polymerase is DNA
polymerase III(DNA Pol III) in prokaryotes and presumably Pol ε in
yeasts.
Lagging strand
The lagging strand is the strand of the template DNA double helix
that is oriented so that the replication fork moves along it in a 5' to 3'
manner.

15. On the lagging strand, primase "reads" the DNA and adds RNA
to it in short, separated segments. In eukaryotes, primase is
intrinsic to Pol α.
 DNA polymerase III or Pol δ lengthens the primed segments,
forming Okazaki fragments.

16. Regulation
Within eukaryotes, DNA replication is controlled within the
context of the cell cycle
DNA replication occurs during the S phase (synthesis phase).
The G1/S checkpoint regulates whether eukaryotic cells enter
the process of DNA replication and subsequent division.
 Cells that do not proceed through this checkpoint remain in the
G0 stage and do not replicate their DNA.

17. Termination
Eukaryotes initiate DNA replication at multiple points in the
chromosome, so replication forks meet and terminate at many
points in the chromosome; these are not known to be regulated
in any particular way.
Because eukaryotes have linear chromosomes, DNA replication
is unable to reach the very end of the chromosomes, but ends at
the telomere region of repetitive DNA close to the end.
This is a normal process in somatic cells. As a result, cells can
only divide a certain number of times before the DNA loss
prevents further division.
Within the germ cell line, which passes DNA to the next
generation, telomerase extends the repetitive sequences of the
telomere region to prevent degradation. Telomerase can
become mistakenly active in somatic cells, sometimes leading
to cancer formation.

18. Conclusion
DNA replication is semi conservative, with each existing strand
serving as a template for the synthesis of a new strand.
Replication begins at specific locations called origins of
replication. Replication requires a primer.

19. References
Cell &Molecular Biology - Jerald Carp
Biotechnology - U Satyanarayana
Internet- http://en.wikipedia.org/wiki/DNA_replication
http://www.ndsu.edu/pubweb/~mcclean/plsc431/eukarych
rom/eukaryo3.htm