dna replication section 4.3 page 217 why do we need to replicate our dna? when does dna replication...
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DNA ReplicationDNA ReplicationSection 4.3Page 217
Why do we need to replicate our DNA?When does DNA replication occur in a cell?
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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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