A LECTURE ON DNA REPLICATION

1. DNA Replication
Sanju kaladharan

3. How DNA is Packaged (Advanced) - YouTube
(360p).mp4

4. 4
Replication Facts
• DNA has to be copied
before a cell divides
• DNA is copied during the S
or synthesis phase of
interphase
• New cells will need identical
DNA strands
copyright cmassengale

5. 5
Synthesis Phase (S phase)
• S phase during interphase of the
cell cycle
• Nucleus of eukaryotes
Mitosis
-prophase
-metaphase
-anaphase
-telophase
G1 G2
S
phase
interphase
DNA replication takes
place in the S phase.
copyright cmassengale

6. Models of replication
• Semi-conservative replication. In this model, the two strands of DNA
unwind from each other, and each acts as a template for synthesis of a
new, complementary strand. This results in two DNA molecules with one
original strand and one new strand.
• Conservative replication. In this model, DNA replication results in one
molecule that consists of both original DNA strands (identical to the
original DNA molecule) and another molecule that consists of two new
strands (with exactly the same sequences as the original molecule).
• Dispersive replication. In the dispersive model, DNA replication results in
two DNA molecules that are mixtures, or “hybrids,” of parental and
daughter DNA. In this model, each individual strand is a patchwork of
original and new DNA.

7. • DNA replication is semi
conservative, meaning that
each strand in the DNA double
helix acts as a template for the
synthesis of a new,
complementary strand.
• This process takes us from one
starting molecule to two
"daughter" molecules, with
each newly formed double
helix containing one new and
one old strand.

8. The Meselson-Stahl experiment

11. DNA polymerase
• They add nucleotides one by one to
the growing DNA chain, incorporating
only those that are complementary
to the template.
• The addition of nucleotides requires
energy. This energy comes from the
nucleotides themselves, which have
three phosphates attached to them
(much like the energy-carrying
molecule ATP). When the bond
between phosphates is broken, the
energy released is used to form a
bond between the incoming
nucleotide and the growing chain.
Key features
• They always need a template
• They can only add nucleotides to
the 3' end of a DNA strand
• They can't start making a DNA
chain from scratch, but require a
pre-existing chain or short stretch
of nucleotides called a primer
• They proofread, or check their
work, removing the vast majority
of "wrong" nucleotides that are
accidentally added to the chain

12. Prokaryotic and Eukaryotic DNA
polymerases

13. 13
DNA Replication
• Begins at Origins of Replication
• Two strands open forming Replication
Forks (Y-shaped region)
• New strands grow at the forks
Replication
Fork
Parental DNA Molecule
3’
5’
3’
5’copyright cmassengale

14. DNA Replication
• As the 2 DNA strands open at the origin,
Replication Bubbles form
• Prokaryotes (bacteria) have a single bubble
• Eukaryotic chromosomes have MANY
bubbles
copyright cmassengale 14

15. Prokaryotes Eukaryotes

16. 16
DNA Replication
• Enzyme Helicase unwinds and separates
the 2 DNA strands by breaking the weak
hydrogen bonds
• Single-Strand Binding Proteins attach
and keep the 2 DNA strands separated
and untwisted

17. • DNA Replication _ Interactive Easy To
Understand Animation and Discription. -
YouTube (360p).mp4

18. Primers and primases
• DNA polymerases can only add nucleotides to the 3' end of an existing
DNA strand. (They use the free -OH group found at the 3' end as a "hook,"
adding a nucleotide to this group in the polymerization reaction.)
• Primase makes an RNA primer, or short stretch of nucleic acid
complementary to the template, that provides a 3' end for DNA
polymerase to work on.
• A typical primer is about five to ten nucleotides long. The
primer primes DNA synthesis, i.e., gets it started.
• Once the RNA primer is in place, DNA polymerase "extends" it, adding
nucleotides one by one to make a new DNA strand that's complementary
to the template strand.

19. Leading and lagging strands
• In E. coli, the DNA polymerase that handles most of the synthesis is DNA
polymerase III. There are two molecules of DNA polymerase III at a
replication fork, each of them hard at work on one of the two new DNA
strands.
• DNA polymerases can only make DNA in the 5' to 3' direction, and this
poses a problem during replication.
• A DNA double helix is always anti-parallel; in other words, one strand runs
in the 5' to 3' direction, while the other runs in the 3' to 5' direction.
• This makes it necessary for the two new strands, which are also
antiparallel to their templates, to be made in slightly different ways.

20. Leading and lagging strands
• One new strand, which runs 5' to 3'
towards the replication fork, is the easy
one. This strand is made continuously,
because the DNA polymerase is moving
in the same direction as the replication
fork. This continuously synthesized
strand is called the leading strand.
• The other new strand, which runs 5' to
3' away from the fork, is trickier. This
strand is made in fragments because, as
the fork moves forward, the DNA
polymerase (which is moving away from
the fork) must come off and reattach on
the newly exposed DNA. This tricky
strand, which is made in fragments, is
called the lagging strand.
The small fragments are
called Okazaki fragments, named
for the Japanese scientist who
discovered them. The leading
strand can be extended from one
primer alone, whereas the lagging
strand needs a new primer for
each of the short Okazaki
fragments.

21. Other proteins and enzymes involved
• The sliding clamp, which holds DNA polymerase III molecules in place as
they synthesize DNA. The sliding clamp is a ring-shaped protein and keeps
the DNA polymerase of the lagging strand from floating off when it re-
starts at a new Okazaki fragment
• Topoisomerase also plays an important maintenance role during DNA
replication. This enzyme prevents the DNA double helix ahead of the
replication fork from getting too tightly wound as the DNA is opened up. It
acts by making temporary nicks in the helix to release the tension, then
sealing the nicks to avoid permanent damage.
• The RNA primers are removed and replaced by DNA through the activity
of DNA polymerase I, the other polymerase involved in replication. The
nicks that remain after the primers are replaced get sealed by the
enzymeDNA ligase.

22. • Helicase opens up the DNA at the
replication fork.
• Single-strand binding proteins coat
the DNA around the replication fork
to prevent rewinding of the DNA.
• Topoisomerase works at the region
ahead of the replication fork to
prevent supercoiling.
• Primase synthesizes RNA primers
complementary to the DNA strand.
• DNA polymerase III extends the
primers, adding on to the 3' end, to
make the bulk of the new DNA.
• RNA primers are removed and
replaced with DNA by DNA
polymerase I.
• The gaps between DNA fragments
are sealed by DNA ligase.

23. In eukaryotes
• Eukaryotes usually have multiple linear chromosomes, each with multiple
origins of replication. Humans can have up to 100,100,100, origins of
replication
• Most of the E. coli enzymes have counterparts in eukaryotic DNA
replication, but a single enzyme in E. coli may be represented by multiple
enzymes in eukaryotes. For instance, there are five human DNA
polymerases with important roles in replication
• Most eukaryotic chromosomes are linear. Because of the way the lagging
strand is made, some DNA is lost from the ends of linear chromosomes
(the telomeres) in each round of replication.

24. DNA Proof reading and repair
• replication errors and DNA damage are actually
happening in the cells of our bodies all the time. In
most cases, however, they don’t cause cancer, or even
mutations. That’s because they are usually detected
and fixed by DNA proofreading and repair mechanisms.
Or, if the damage cannot be fixed, the cell will undergo
programmed cell death (apoptosis) to avoid passing on
the faulty DNA.
• Mutations happen, and get passed on to daughter
cells, only when these mechanisms fail. Cancer, in turn,
develops only when multiple mutations in division-
related genes accumulate in the same cell.

25. Proof reading
• During DNA replication
(copying), most DNA
polymerases can “check their
work” with each base that
they add. This process is
called proofreading.
• If the polymerase detects
that a wrong (incorrectly
paired) nucleotide has been
added, it will remove and
replace the nucleotide right
away, before continuing with
DNA synthesis^1

26. Mismatch repair
• Mismatch repair happens right after new DNA has been made, and its job
is to remove and replace mis-paired bases (ones that were not fixed
during proofreading).
• Mismatch repair can also detect and correct small insertions and deletions
that happen when the polymerases "slips," losing its footing on the
template.
• In bacteria, original and newly made strands of DNA can be told apart by a
feature called methylation state. An old DNA strand will have methyl
groups attached to some of its bases, while a newly made DNA strand will
not yet have gotten its methyl group
• In eukaryotes, the processes that allow the original strand to be identified
in mismatch repair involve recognition of nicks (single-stranded breaks)
that are found only in the newly synthesized DNA

27. • First, a protein complex (group of
proteins) recognizes and binds to
the mispaired base.
• A second complex cuts the DNA
near the mismatch, and more
enzymes chop out the incorrect
nucleotide and a surrounding
patch of DNA.
• A DNA polymerase then replaces
the missing section with correct
nucleotides, and an enzyme
called a DNA ligase seals the gap

28. DNA damage repair mechanisms
• Direct reversal: Some DNA-damaging chemical reactions can be directly
"undone" by enzymes in the cell.
• Excision repair: Damage to one or a few bases of DNA is often fixed by
removal (excision) and replacement of the damaged region. In base
excision repair, just the damaged base is removed. In nucleotide excision
repair, as in the mismatch repair we saw above, a patch of nucleotides is
removed.
• Double-stranded break repair: Two major pathways, non-homologous end
joining and homologous recombination, are used to repair double-
stranded breaks in DNA (that is, when an entire chromosome splits into
two pieces).

29. Reversal of damage
• "DNA damage" often just
involves an extra group of atoms
getting attached to DNA through
a chemical reaction.
• For example, guanine (G) can
undergo a reaction that attaches
a methyl group to an oxygen
atom in the base.
• The methyl-bearing guanine, if
not fixed, will pair with thymine
(T) rather than cytosine (C)
during DNA replication. Luckily,
humans and many other
organisms have an enzyme that
can remove the methyl group,
reversing the reaction and
returning the base to normal

30. Base excision repair
• Base excision repair is a mechanism used to detect and remove certain
types of damaged bases. A group of enzymes called glycosylases play a
key role in base excision repair. Each glycosylase detects and removes a
specific kind of damaged base.
• For example, a chemical reaction called deamination can convert a
cytosine base into uracil, a base typically found only in RNA. During DNA
replication, uracil will pair with adenine rather than guanine (as it would if
the base was still cytosine), so an uncorrected cytosine-to-uracil change
can lead to a mutation
• To prevent such mutations, a glycosylase from the base excision repair
pathway detects and removes deaminated cytosines. Once the base has
been removed, the "empty" piece of DNA backbone is also removed, and
the gap is filled and sealed by other enzymes

32. Nucleotide excision repair
• Nucleotide excision repair is also used to fix some types of damage caused
by UV radiation, for instance, when you get a sunburn.
• UV radiation can make cytosine and thymine bases react with neighboring
bases that are also Cs or Ts, forming bonds that distort the double helix
and cause errors in DNA replication.
• The most common type of linkage, a thymine dimer, consists of two
thymine bases.
• In nucleotide excision repair, the damaged nucleotide(s) are removed
along with a surrounding patch of DNA. In this process, a helicase (DNA-
opening enzyme) cranks open the DNA to form a bubble, and DNA-
cutting enzymes chop out the damaged part of the bubble.
• A DNA polymerase replaces the missing DNA, and a DNA ligase seals the
gap in the backbone of the strand that react with each other and become
chemically linked

34. Double-stranded breaks repair
• Double-stranded breaks are dangerous because large segments of
chromosomes, and the hundreds of genes they contain, may be lost if the
break is not repaired. Two pathways involved in the repair of double-
stranded DNA breaks are the non-homologous end joining and
homologous recombination pathways.

35. Non-homologous end joining
• In non-homologous end joining,
the two broken ends of the
chromosome are simply glued
back together.
• This repair mechanism is “messy”
and typically involves the loss, or
sometimes addition, of a few
nucleotides at the cut site.
• So, non-homologous end joining
tends to produce a mutation, but
this is better than the alternative
(loss of an entire chromosome
arm)

36. Homologous recombination
• In homologous recombination,
information from the homologous
chromosome that matches the
damaged one (or from a sister
chromatid, if the DNA has been
copied) is used to repair the break.
• In this process, the two homologous
chromosomes come together, and
the undamaged region of the
homologue or chromatid is used as a
template to replace the damaged
region of the broken chromosome.
• Homologous recombination is
“cleaner” than non-homologous end
joining and does not usually cause
mutations^{11}

38. TELOMERES AND TELOMERASES
• Repetitive regions at the very ends of chromosomes are called telomeres,
and they're found in a wide range of eukaryotic species, from human
beings to unicellular protists.
• Telomeres act as caps that protect the internal regions of the
chromosomes, and they're worn down a small amount in each round of
DNA replication.
• Unlike bacterial chromosomes, the chromosomes of eukaryotes are linear
(rod-shaped), meaning that they have ends.
• These ends pose a problem for DNA replication. The DNA at the very end
of the chromosome cannot be fully copied in each round of replication,
resulting in a slow, gradual shortening of the chromosome.

39. • In most cases, the primers of the Okazaki fragments can be easily
replaced with DNA and the fragments connected to form an
unbroken strand.
• When the replication fork reaches the end of the chromosome,
however, there is (in many species, including humans) a short
stretch of DNA that does not get covered by an Okazaki fragment—
essentially, there's no way to get the fragment started because the
primer would fall beyond the chromosome end.
• Also, the primer of the last Okazaki fragment that does get made
can't be replaced with DNA like other primers.

40. • A part of the DNA at the end of a eukaryotic chromosome goes
uncopied in each round of replication, leaving a single-stranded
overhang.
• Over multiple rounds of cell division, the chromosome will get
shorter and shorter as this process repeats.
• In human cells, the last RNA primer of the lagging strand may be
positioned as much as 70 to 100 nucleotides away from the
chromosome.
• Thus, the single-stranded overhangs produced by incomplete end
replication in humans are fairly long, and the chromosome shortens
significantly with each round of cell division.

42. • To prevent the loss of genes as chromosome ends wear down, the tips of
eukaryotic chromosomes have specialized DNA “caps” called telomeres.
• Telomeres consist of hundreds or thousands of repeats of the same short
DNA sequence, which varies between organisms but is 5'-TTAGGG-3' in
humans and other mammals.
• Telomeres need to be protected from a cell's DNA repair systems because
they have single-stranded overhangs, which "look like" damaged DNA. The
overhang at the lagging strand end of the chromosome is due to
incomplete end replication.
• The overhang at the leading strand end of the chromosome is actually
generated by enzymes that cut away part of the DNA.
• In some species (including humans), the single-stranded overhangs bind to
complementary repeats in the nearby double-stranded DNA, causing the
telomere ends to form protective loops.
• Proteins associated with the telomere ends also help protect them and
prevent them from triggering DNA repair pathways.

43. • The repeats that make up a
telomere are eaten away slowly
over many division cycles,
providing a buffer that protects
the internal chromosome regions
bearing the genes (at least, for
some period of time).
• Telomere shortening has been
connected to the aging of cells,
and the progressive loss of
telomeres may explain why cells
can only divide a certain number
of times

44. Telomerase
• Some cells have the ability to reverse telomere shortening by
expressing telomerase, an enzyme that extends the telomeres of
chromosomes.
• Telomerase is an RNA-dependent DNA polymerase,
• The enzyme binds to a special RNA molecule that contains a sequence
complementary to the telomeric repeat.
• It extends (adds nucleotides to) the overhanging strand of the telomere
DNA using this complementary RNA as a template.
• When the overhang is long enough, a matching strand can be made by
the normal DNA replication machinery (that is, using an RNA primer and
DNA polymerase), producing double-stranded DNA.
• The primer may not be positioned right at the chromosome end and
cannot be replaced with DNA, so an overhang will still be present.
However, the overall length of the telomere will be greater

46. • Telomerase is not usually active in most somatic cells (cells of the body),
but it’s active in germ cells (the cells that make sperm and eggs) and some
adult stem cells.
• These are cell types that need to undergo many divisions, or, in the case
of germ cells, give rise to a new organism with its telomeric “clock” reset.
• Interestingly, many cancer cells have shortened telomeres, and
telomerase is active in these cells.
• If telomerase could be inhibited by drugs as part of cancer therapy, their
excess division (and thus, the growth of the cancerous tumor) could
potentially be stopped.

50. Thank you