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DNA REPLICATION. YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE THE DEPARTMENT OF MEDICAL BIOLOGY ASST. PROF. DR. ENDER ŞİMŞEK. DNA Replication. Process of duplication of the entire genome prior to cell division. DNA Replication.

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dna replication

DNA REPLICATION

YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE

THE DEPARTMENT OF MEDICAL BIOLOGY

ASST. PROF. DR. ENDER ŞİMŞEK

dna replication1
DNA Replication

Process of duplication of the entire genome

prior to cell division.

dna replication2
DNA Replication

In eukaryotes , replication only occurs during the S phaseof the cell cycle and replication rate is slower resulting in a higher accuracy of replication.

the mechanism of dna replication
The mechanism of DNA replication
  • Three Important Steps of DNA Replication:

1. Initiation

2. Elongation

3. Termination

the mechanism of dna replication1
The mechanism of DNA replication
  • Initiation
    • Proteins bind to DNA and open up double helix
    • Prepare DNA for complementary base pairing
the mechanism of dna replication2
The mechanism of DNA replication
  • Initiation
    • Proteins bind to DNA and open up double helix
    • Prepare DNA for complementary base pairing
  • Elongation
    • Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA
the mechanism of dna replication3
The mechanism of DNA replication
  • Initiation
    • Proteins bind to DNA and open up double helix
    • Prepare DNA for complementary base pairing
  • Elongation
    • Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA
  • Termination
    • Proteins release the replication complex
features of dna replication
Features of DNA Replication
  • DNA replication is semiconservative
    • Each strand of template DNA is being copied.
features of dna replication1
Features of DNA Replication
  • DNA replication is semiconservative
    • Each strand of template DNA is being copied.
  • DNA replication is semidiscontinuous
    • The leading strand copies continuously
    • The lagging strand copies in segments which must be joined
features of dna replication2
Features of DNA Replication
  • DNA replication is semiconservative
    • Each strand of template DNA is being copied.
  • DNA replication is semidiscontinuous
    • The leading strand copies continuously
    • The lagging strand copies in segments which must be joined
  • DNA replication is bidirectional
    • Bidirectional replication involves two replication forks, which move in opposite directions
flow of genetic information in the cell
Flow of Genetic Information in the Cell
  • Mechanisms by which information is transferred in the cell is based on “Central Dogma”
proposed models of dna replication
Proposed Models of DNA Replication
  • In the late 1950s, three different mechanisms were proposed for the replication of DNA;
proposed models of dna replication1
Proposed Models of DNA Replication
  • In the late 1950s, three different mechanisms were proposed for the replication of DNA;
    • Conservative model
      • Both parental strands stay together after DNA replication
proposed models of dna replication2
Proposed Models of DNA Replication
  • In the late 1950s, three different mechanisms were proposed for the replication of DNA;
    • Conservative model
      • Both parental strands stay together after DNA replication
    • Semiconservative model
      • The double-stranded DNA contains one parental and one daughter strand following DNA replication
proposed models of dna replication3
Proposed Models of DNA Replication
  • In the late 1950s, three different mechanisms were proposed for the replication of DNA;
    • Conservative model
      • Both parental strands stay together after DNA replication
    • Semiconservative model
      • The double-stranded DNA contains one parental and one daughter strand following DNA replication
    • Dispersive model
      • Parental and daughter DNA are interspersed in both strands following DNA replication
three models for dna replication
Three Models for DNA Replication

Semiconservative

  • Both parental strands stay together after DNA replication

Conservative

  • The double-stranded DNA contains one parental and
  • one daughter strand following DNA replication

Dispersive

  • Parental and daughter DNA are interspersed in both strands following DNA replication
how is dna replicated
How Is DNA Replicated?

Meselson and Stahl showed semiconservative replication was the correct model for the replication of DNA.

They used density labeling to distinguish parent DNA strands from new DNA strands.

DNA was labeled with 15N, making DNA more dense.

slide18
Matthew Meselson and Franklin Stahl experiment in 1958
    • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations (The cells get heavy-labeled DNA).
slide19
Matthew Meselson and Franklin Stahl experiment in 1958
    • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations (The cells get heavy-labeled DNA).
    • Switch to medium containing only 14N (a light isotope of Nitrogen).
slide20
Matthew Meselson and Franklin Stahl experiment in 1958
    • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations (The cells get heavy-labeled DNA).
    • Switch to medium containing only 14N (a light isotope of Nitrogen).
    • Collect sample of cells after various times.
slide21
Matthew Meselson and Franklin Stahl experiment in 1958
    • Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations (The cells get heavy-labeled DNA)
    • Switch to medium containing only 14N (a light isotope of Nitrogen)
    • Collect sample of cells after various times
    • Analyze the density of the DNA by centrifugation using a CsClgradient.
the meselson stahl experiment part 2
The Meselson- Stahl Experiment (Part 2)

2ND GENERATION

1ST GENERATION

slide24

DNA

14N

15N

CsCl Density Gradient Centrifugation

slide25

Interpreting the Data

After one generation, DNA is “half-heavy”

After ~ two generations, DNA is of two types:

“light” and “half-heavy”

This is consistent with only the semi-conservative model

how is dna replicated1
How Is DNA Replicated?

Results of their experiment explained by the semiconservative model:

If conservative, the first generation of individuals would have been both high and low density, but not intermediate.

how is dna replicated2
How Is DNA Replicated?

Results of their experiment explained by the semiconservative model:

If conservative, the first generation of individuals would have been both high and low density, but not intermediate.

If dispersive, density in the first generation would be half, but this density would not appear in subsequent generations.

slide28

1955: Arthur Kornberg

  • Worked with E. coli.
  • Discovered the mechanisms of DNA synthesis.
  • Components are required:
  • dNTPs: dATP, dTTP, dGTP, dCTP

(deoxyribonucleoside 5’-triphosphates)

(sugar-base + 3 phosphates)

2. DNA template

  • DNA polymerase I (Kornberg enzyme)
  • Mg 2+ (optimizes DNA polymerase activity)
  • PRIMERS!!!
how is dna replicated3
How Is DNA Replicated?

Two steps in DNA replication:

The double helix is unwound, making two template strands.

how is dna replicated4
How Is DNA Replicated?

Two steps in DNA replication:

The double helix is unwound, making two template strands.

New nucleotides are added to the new strand at the 3′ end and joined by phospho-diesterlinkages. (Sequence is determined by complementary base pairing.)

each new dna strand grows from its 5 end to its 3 end
Each New DNA Strand Grows from Its 5´ End to Its 3´ End

New nucleotides are added

to the new strand at the 3′ end

and joined by phospho-diester

linkages.

each new dna strand grows from its 5 end to its 3 end1
Each New DNA Strand Grows from Its 5´ End to Its 3´ End

DNA chain elongation catalyzed

by DNA polymerase

how is dna replicated5
How Is DNA Replicated?

All chromosomes have a region called origin of replication (ori).

Proteins in the replication complex bind to a DNA sequence in ori.

how is dna replicated6
How Is DNA Replicated?

*** DNA replication begins with a short primer— a starter strand.

*** The primer is complementary to the DNA template.

*** Primase—an enzyme—synthesizes DNA one nucleotide at a time.

*** DNA polymerase adds nucleotides to the 3′ end.

dna polymerase binds to the template strand
DNA Polymerase Binds to the Template Strand

***DNA polymerases are larger than their substrates, the dNTPs, and the template DNA.

***The enzyme is shaped like an open right hand—the “palm” brings the active site and the substrates into contact.

***The “fingers” recognize the nucleotide bases.

how is dna replicated8
How Is DNA Replicated?

Other proteins play a role in replication as well;

DNA helicaseuses energy from ATP hydrolysis to unwind the DNA.

Single-strand DNA binding proteins keep the strands from getting back together.

how is dna replicated9
How Is DNA Replicated?

The replication fork is the site where DNA unwinds to expose bases.

One new strand, the leading strand, is oriented to grow at its 3′ end as the fork opens.

The lagging strand is oriented so that its exposed 3′ end gets farther from the fork.

how is dna replicated10
How Is DNA Replicated?

Synthesis of the lagging strand occurs in small, discontinuous stretches—Okazaki fragments.

Each Okazaki fragment requires its own primer, synthesized by the primase.

DNA polymerase III adds nucleotides to the 3′ end, until reaching the primer of the previous fragment.

how is dna replicated11
How Is DNA Replicated?

DNA polymerase I then replaces the primer with DNA.

The final phospho-diesterlinkage between fragments is catalyzed by DNA ligase.

how is dna replicated12
How Is DNA Replicated?

Small, circular chromosomes have a single origin of replication.

As DNA moves through the replication complex, two interlocking circular chromosomes are formed.

DNA topoisomerase separates the two chromosomes.

how is dna replicated13
How Is DNA Replicated?

Newly replicated strand is stabilized by a sliding DNA clamp (a protein).

The sliding DNA clamp was recognized in dividing cellscalled the proliferating cell nuclear antigen (PCNA).

slide46

Eukaryotic Enzymes:

Five common DNA polymerases from mammals.

Polymerase  (alpha): nuclear, DNA replication, no proofreading

Polymerase  (beta): nuclear, DNA repair, no proofreading

Polymerase  (gamma): mitochondria, DNA replication, proofreading

Polymerase  (delta): nuclear, DNA replication, proofreading

Polymerase  (epsilon): nuclear, DNA repair (?), proofreading

how is dna replicated14
How Is DNA Replicated?

Large linear chromosomes have many origins of replication.

Replication complexes bind to the sites at the same time and catalyze simultaneous replication.

slide48

Origin of replication (e.g., the prokaryote example):

  • Begins with double-helix denaturing into single-strands thus exposing the bases.
  • Exposes a replication bubble from which replication proceeds in both directions.

~245 bp in E. coli

slide53

What about the ends (or telomeres) of linear chromosomes?

DNA polymerase/ligase cannot fill the gap at end of chromosome after RNA primer is removed.

fig 3 14 the problem of replicating completely a linear chromosome in eukaryotes
Fig. 3.14 The problem of replicating completely a linear chromosome in eukaryotes

DNA polymerase/ligase cannot fill the gap at end of chromosome after RNA primer is removed.

If this gap is not filled, chromosomes would become shorter each round of replication!

slide55

TelomereandTelomerase

Telomerase add TTGGGG sequences to the end of chromosome to prevent the shortening of the telomeres in each replication.

slide56

TelomereandTelomerase

Added sequences forms “the hairpin structure” and between G bases H bond is established and the free 3'-OH group is obtained.

slide57

TelomereandTelomerase

After DNA pol I fills the gap, the Hairpin structure removed and finally the DNA loss is prevented.

slide58

Model for the “replication machine,” or replisome, the complex of key replication proteins, with the DNA at the replication fork