Sinh học tế bào và gene

Sao chép DNA và sửa chữa DNA

TS. Lê Quang Dũng
Bộ môn Sinh học Tế bào
Email: lequangdung@hus.edu.vn

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1. What Are Genes Made Of

What are genes made of ? DNA or protein?

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- In 1952 Alfred Hershey and Martha Chase started to work on this question.
- T2 virus infections begin when the virus attaches to the cell wall of E. coli and infects the
host cells.
- T2 is made up of both protein and DNA.

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- Proteins contain sulfur, and DNA contains phosphorus.

-In order to differ protein and DNA, viruses were grown in the presence of either a
radioactive isotope of sulfur (35S) or a radioactive isotope of phosphorus (32P).

- This step produced a population of viruses with radioactive proteins and a population
with radioactive DNA.

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- Each set of radioactive viruses was used to infect E. coli cells.

- If genes consist of DNA, then radioactive protein should be found only in the capsids
outside the infected host cells, while radioactive DNA should be located inside the cells.

- If genes consist of proteins, then radioactive protein and no radioactive DNA should be
inside the cells.

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-The virus capsids were sheared off the cells using kitchen blenders.

- The result shows that the radioactive protein was outside cells in the emptied capsids,
while virtually all the radioactive DNA was inside the host cells.

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 The Secondary Structure of DNA

-In 1953, Watson and Crick proposed a model for the secondary structure of DNA.

-DNA is typically double-stranded with each strand consisting of a long, linear polymer
made up of monomers called deoxyribonucleotides.

- Each deoxyribonucleotide consists of a deoxyribose sugar, a phosphate group, and a

nitrogenous base.

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-Deoxyribonucleotides link together by phosphodiester bon between a hydroxyl group
on the 3′ and a phosphate group attached to the 5′ carbon.

- The primary structure of each strand of DNA has two major features: a “backbone”
made up of the deoxyribonucleotide monomers, and a series of bases that project from
the backbone.

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- Two of antiparallel long strands in opposite directions forms double helix or double-
stranded Spiral structure.

- The two strands are linked together by hydrogen bonds following complementary base
pairing.

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 2. Testing Early Hypotheses about DNA
Synthesis

- Complementary base pairing provided a basis for copying DNA, but it did not reveal a
mechanism. How was DNA replicated?
- If they could tag parental and daughter strands of DNA in a way that would make them
distinguishable from each other, they could determine whether replication was
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conservative, semiconservative, or dispersive.
3. A Model for DNA Synthesis

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- DNA polymerase catalyzes DNA synthesis.
- DNA synthesis always proceeds in the 5′ to 3′ direction.
- deoxyribonucleoside triphosphates (dNTPs) have high potential energy due to their three
phosphate groups. This is enough to provide energy for DNA synthesis.

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 Where Does Replication Start
- A replication bubble forms when DNA is being synthesized. Initially, the replication
bubble forms at a specific sequence of bases called the origin of replication.
- Bacterial chromosomes have only one origin of replication, while eukaryotes have multiple
origins of replication along each chromosome.
- Active DNA synthesis takes place at the replication forks of each replication bubble in both
directions at the same time.

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 How Is the Helix Opened and Stabilized?

-In eukaryotes, the proteins that initiate DNA replication are under tight control by cell-cycle
regulatory proteins.

-Once a specific set of proteins recognizes the origin on a bacterial or a eukaryotic

chromosome, the enzyme DNA helicase breaks the hydrogen bonds between the base pairs
in that location and opens the double helix there.

-Single-strand DNA–binding proteins (SSBPs) attach to the separated strands to prevent

them from snapping back into a double helix.

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- The unwinding of DNA strands at the replication fork creates twists farther down the
helix which is relaxed by proteins called topoisomerases.

- A topoisomerase is an enzyme that cuts DNA, allows it to unwind, and rejoins it ahead
of the advancing replication fork.

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 How Is the Leading Strand Synthesized?

-DNA polymerase works only in the 5′ S 3′ direction along a single-stranded template.

- This enzyme requires a 3′ primer.

- These restrictions control how synthesis occurs on both template strands of DNA.

- The single-stranded template dictates which deoxyribonucleotide should be added next.

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-Before DNA synthesis can begin, an enzyme called primase synthesizes the short stretch
of RNA that acts as a primer for DNA polymerase.

- Primase is one type of RNA polymerase—a class of enzymes that catalyze the
polymerization of ribonucleotides into RNA.

- Unlike DNA polymerases, primase and other RNA polymerases do not require a primer
to begin synthesis.

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-The strand of DNA that is synthesized toward the replication fork is called the leading
strand, or continuous strand.

- Once a primer has bound to the template, DNA polymerase begins adding
deoxyribonucleotides in the 5′ S 3′ direction.

- Part of DNA polymerase forms a ring—the sliding clamp—that surrounds the DNA, and
another part grips the DNA strand in a way that’s similar to your hand clasping a rope.

- Deoxyribonucleotide addition is catalyzed at an active site in a groove between the

enzyme’s “thumb” and “fingers.”

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 How Is the Lagging Strand Synthesized?

-The two strands of the DNA double helix run in opposite directions (they’re
antiparallel) but that DNA polymerase operates only in the 5′ to 3′ direction.
- The 5’ to 3’ strand, appropriately called the lagging strand, or discontinuous strand,
synthesized in a direction that runs away from the moving replication fork, and it leaves
gaps between single-stranded templates DNA.

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 The Discovery of Okazaki Fragments

- When DNAs are purified from the cells, separated the two strands of DNA, and
analyzed the short size of the labeled molecules.
- These short DNAs attached to RNA primers came to be known as Okazaki fragments.
- The short DNAs were labeled during the pulse, and they gradually became longer
during the chase. This is because Okazaki fragments are linked together soon after
they’re formed.

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- DNA polymerase I attaches to the 3′ end of an Okazaki fragment.

- As DNA polymerase I moves in the 5′ to 3′ direction, it removes the RNA primer ahead
of it and replaces the ribonucleotides with deoxyribonucleotides.

- Once the RNA primer is removed and replaced by DNA, enzyme DNA ligase catalyzes
the formation of a phosphodiester bond between the adjacent fragments to fill in the
gaps.

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Proteins Required for DNA Synthesis in Bacteria

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 4. Replicating the Ends of Linear Chromosomes

-Replication at the very ends of eukaryotic chromosomes called telomere is

complicated. Replication of chromosome ends requires a special enzyme to complete.
- When the replication fork reaches the end, a DNA polymerase synthesizes the leading
strand all the way to the end of the parent DNA template.
- On the lagging strand, primase adds an RNA primer close to the end of the
chromosome.

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- DNA polymerase synthesizes the final Okazaki fragment of the lagging strand. An enzyme
that degrades ribonucleotides removes the primer.

- DNA polymerase is unable to add DNA near the end as it cannot synthesize DNA without
a primer, leaving the lagging strand stays single-stranded.

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 Telomerase Solves the End Replication Problem
- Telomeres are made of short stretches of bases that are repeated over and over.
- An enzyme called telomerase that carries its own template is involved in replicating
telomeres.
-Telomerase is extraordinary because it catalyzes the synthesis of DNA from an RNA
template that it contains, preventing it from getting shorter.

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 5. Repairing Mistakes and DNA Damage

- DNA replication must be accurate, allowing an error rate about one mistake per billion
deoxyribonucleotides.

- Humans develop from a fertilized egg that has roughly 12 billion deoxyribonucleotides
in its DNA.

-This DNA is replicated over and over to create the trillions of cells that eventually make
up the adult body.

- Therefore, the accurate replication of DNA is a matter of life and death.

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 Correcting Mistakes in DNA Synthesis

-DNA Polymerase proofreads the newly synthesized DNA using a combination of enzymes.
- DNA polymerase’s active site can discriminate between these shapes to identify the
mismatched base. It only add a new deoxyribonucleotide only when the previous base
pair is correct.
- The exonuclease activity of the DNA polymerase III ε subunit then removes the
mismatched deoxyribonucleotide.

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 Mismatch Repair

-Occasionally, DNA polymerase leaves a mismatched base behind in the newly

synthesized strand.

- Once DNA synthesis moves beyond a mismatched base pair, proofreading is no longer
possible. This will require mismatch repair mechanism, a form of error correction.

-There are 10 E. coli proteins identified so far involved in different aspects of mismatch
repair.

- These proteins recognize the mismatch, remove a section of the DNA strand that
includes the incorrect base, and then resynthesize the missing DNA using the older
strand as a template.

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 Repairing Damaged DNA

- Genes can be damaged by many factors. To fix damaged DNA, organisms have evolved
a wide array of DNA damage-repair systems.
-An example is the nucleotide excision repair system that works on DNA damage caused
by ultraviolet light and many different chemicals.
- Ultraviolet (UV) light in sunlight can cause a covalent bond to form between adjacent
pyrimidine bases within the same DNA strand.

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-Nucleotide excision repair removes many types of damage in the DNA helix.

- In the first step of excision repair, a protein complex recognizes the kink in the DNA
helix. Once a damaged region is recognized, enzymes remove a segment of single
stranded DNA containing the defective sequence.

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- The intact DNA strand provides a template for synthesis of a corrected strand. The 3′
hydroxyl of the DNA strand next to the gap serves as a primer.

- DNA ligase links the newly synthesized DNA to the original undamaged DNA. As with
mismatch repair, multiple enzymes work together and DNA synthesis plays a central role in
repair.

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