DNA Replication Section 4.3 Page 217 Why do we need to replicate our DNA? When does DNA replication...

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DNA Replication DNA Replication Section 4.3 Page 217 Why do we need to replicate our DNA? When does DNA replication occur in a cell?

Transcript of DNA Replication Section 4.3 Page 217 Why do we need to replicate our DNA? When does DNA replication...

Page 1: DNA Replication Section 4.3 Page 217 Why do we need to replicate our DNA? When does DNA replication occur in a cell?

DNA ReplicationDNA ReplicationSection 4.3Page 217

Why do we need to replicate our DNA?When does DNA replication occur in a cell?

Page 2: DNA Replication Section 4.3 Page 217 Why do we need to replicate our DNA? When does DNA replication occur in a cell?

BackgroundBackgroundCell division: mitosis + cytokinesis

DNA replicated in interphase, prior to mitosis

Each daughter cell must have an exact copy of the parent cell’s DNA

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But how does replication occur? But how does replication occur? Scientists in the 50s had 3 proposed Scientists in the 50s had 3 proposed models:models:

1. Semi-conservative2. Conservative3. Dispersive

But which one is right???

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Semi-conservative – Two parental strands separate and each serves as a template for a new progeny strand.

Newly-synthesized DNA molecules has one old strand,and one new strand

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Conservative – the two parental strands stay together, and somehow produce another daughter helix with completely new strands.

Newly-synthesized DNA molecules has two new strands.

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Dispersive – DNA becomes fragmented so that new and old DNA coexist in the same strand after replication.

Newly-synthesized DNA molecule has pieces of oldand new strands interspersed.

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Meselson-Stahl experiment, 1958Meselson-Stahl experiment, 1958

Purpose: to elucidate the mode of replication

Experimental model: E. coli

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Grew E. coli in a medium enriched with a heavy nitrogen isotope (15N)◦Denser-than-normal DNA

Switched cells to ordinary medium with 14N, and allowed DNA replication

Will 14N be incorporated into DNA strands with 15N?

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Centrifuged the DNA within a density gradient: Separates components according to density

*A centrifuge is a device that spins a solution at high speeds, the spinning splits up the different components in a mixture based on density

http://highered.mcgraw-hill.com/olc/dl/120076/bio22.swf

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Possible results:

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Observed:First generation – One intermediate bandSecond generation – One light/one

intermediate

Conclusion: DNA replication is semi-conservative

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DNA REPLICATION:DNA REPLICATION:THE DETAILSTHE DETAILS

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Three stages:Three stages:Stage 1: Initiation◦DNA strands are separated◦A small portion of RNA is annealed to the

exposed strands to “prime” them for replication

Stage 2: Elongation:◦ DNA polymerase III builds a new strand of DNA

by incorporating nucleotides

Stage 3: Termination

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Stage 1: InitiationStage 1: Initiation

Separation of strands:

DNA strands are “unzipped” by DNA helicase◦Hydrogen bonds between complementary bases

are broken

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Single-stranded binding proteins (SSBs) bind to exposed strands to prevent re-annealing of strands.

DNA gyrase relieves torsion tension by cutting and re-annealing the two strands

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Priming:

DNA polymerase cannot start incorporating nucleotides on its own◦Needs an existing 3’ end of a nucleic acid

A short segment of RNA (a “primer” – 10 to 60 nucleotides long) provides that 3’ end

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RNA Primase synthesizes the primer and anneals it to the template strand.

DNA polymerase can then add on DNA nucleotides

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Stage 2: ElongationStage 2: ElongationNew strand is synthesized in the 5’ to 3’ direction

(added on to the end with the -OH group)

Catalyzed by DNA polymerase III

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Free bases are floating in the nucleoplasm as deoxyribonucleoside triphosphates.

Energy required for DNA synthesis is provided by hydrolyzing the bond between the 1st and 2nd phosphates

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Characteristics of elongation:Characteristics of elongation:

A. Bi-directionalityB. Semi-discontinuity

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A. Elongation is bi-directional

Elongation proceeds in two directions, outwards from the origin of replication.

The junction where the strands are still joined is called the replication fork.

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DNA synthesis occurs simultaneously using both strands as templates◦ A replication bubble forms between two replication

forks

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B. Elongation is semi-discontinuous

DNA synthesis always occurs in the 5’ to 3’ direction (of the new strand!)

The two template strands are antiparallel Only one strand can be built continuously

http://highered.mcgraw-hill.com/olc/dl/120076/micro04.swf

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Leading strand – Uses the 3’ to 5’ template strand as its guide◦ Is built continuously, towards the replication fork

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Lagging strand – Uses the 5’ to 3’ template strand as its guide◦ Is built discontinuously in short fragments

RNA primase constantly adds new RNA primers along the template strand.

The fragments are called Okazaki fragments.

= site of new primer

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Removal of the RNA primers, and joining of the Okazaki fragments:

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Enzyme Role

DNA polymerase I removes the RNA primers;

replaces them with the proper deoxyribonucleosides

DNA ligase joins the fragments together (phosphodiester bonds)

Removal of the RNA primers, and joining of the Okazaki fragments:

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Stage 3: TerminationStage 3: TerminationTwo replication forks meet each other; orDNA Polymerase III reaches the end of a

strand

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Problem: Shortening of telomeres

Telomeres: The ends of DNA. Contain repetitive sequences.

Protects the chromosome from degradation.

Loss of telomeric DNA occurs on the lagging strand with each replication.

http://spine.rutgers.edu/cellbio/assets/flash/tel.htm

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Lagging strand:

No free 3’ end to replace RNA with DNA

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Approximately 50 replications before the telomeres become too short.

Telomere shortening linked to aging.

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TelomeraseTelomeraseTelomerase - enzyme that prevents shortening

of telomeres

Present in cells that need to divide constantly: white blood cells, germ line cells

May be present in cancerous cells

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ProofreadingProofreadingDNA polymerase III and DNA polymerase I

are constantly proofreading the progeny strand as it is synthesized.

Both have exonuclease activity can identify incorrectly added nucleotides, backtrack,

and excise them (cut them out) before continuing synthesis.

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Further proofreading mechanisms are in place when synthesis is completed.

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Recap:Recap:

Three important properties of DNA replication:

1.DNA replication is semi-conservative2.DNA replication is bi-directional3.DNA replication is semi-discontinuous

…recall what these mean!!

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Enzymes/proteins involved in DNA replication:

DNA helicaseDNA gyrasesingle-stranded binding proteinsRNA primaseDNA polymerase IIIDNA polymerase IDNA ligaseDNA telomerase

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