Ch. 12

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DNA Replication

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DNA Replication

process by which a cell doubles its DNA before divison

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What were the 3 proposed models for DNA?

conservative, dispersive, semi-conservative

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Semi-conservative model of DNA Replication

2 nucleotide strands of DNA separate, and each strand serves as a template for a new strand

  • 1 original strand, 1 new strand

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Conservative model of DNA replication

entire double-stranded DNA molecule serves as template for a whole new molecule

  • original double helix and new double helix

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Dispersive model for DNA replication

both nucleotide strands disperse into fragments = templates for new synthesis of new DNA fragments → then reassemble into new DNA molecules

  • original and new DNA fragments mixed, no original strands preserved

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What did the Meselson and Stahl centrifugation experiment prove and how?

  • Grew bacteria in medium of 15N, 15N then one round in 14N, and in just 14N.

  • 15N = heavier isotope

  • DNA of bacteria incorporated their respective nitrogen isotope into their nitrogenous bases

  • Centrifugation revealed:

    • heavy DNA (15N) appeared as single band

    • DNA allowed one round in 14N = single band at intermediate weight

    • 2 rounds in 14N = 2 bands, one light and one intermediate in weight

    • Several rounds of replication = progressively lighter, and 2 bands 1 intermediate, 1 very light.

  • Proved semi-conservative model

    • conservative cannot produce intermediate weight

    • dispersive wouldn’t produce 2 bands, all would be intermediate

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How many bands of DNA would be expected in Meselson and Stahl’s experiment after 2 rounds of conservative replication in 14N medium?

Two bands

  • one heavy and one light

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How can semiconservative replication differ between organisms?

differ in whether template DNA is linear or circular

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Replicon

a segment of DNA that undergoes replication

  • origin of replication to end of replication on either side of origin

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Origin of replication

site/DNA sequence where DNA replication is initiated

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Bacterial mode of replication

  • circular chromosome

  • single origin of replication (ori)

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Eukaryotic mode of replication

  • Linear chromosomes

  • multiple origins of replication

  • ends of linear molecules require special replication

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In what kind of DNA does Theta replication occur?

circular DNA (bacteria)

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What initiates Theta replication and how does it progress?

  • initiated by unwinding of the two nucleotide strands = replication bubble

  • unwinding continuously at one or both ends of bubble = making it larger

  • DNA repliation on both template strands simultaneous with unwinding until 2 replication forks meet

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How did Theta replication get its name?

generates an intermediate structure that resembles Greek letter theta θ.

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In which direction does DNA replication occur?

DNA replication is bidirectional, forming a replication bubble and 2 replication forks

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Replication fork

point at which the 2 strands of DNA are separated to allow replication of each strand

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Who provided the first visible evidence of theta replication and how?

John Cairns

  • grow bacteria in presence of radioactive nucleotides

  • after replication = 1 radioactive strand and 1 nonradioactive

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Rolling-Circle Replication

  • viruses

  • replication of circular DNA

  • initiated by break in one of the nucleotide strands = double-stranded circular DNA + single-stranded circular DNA

  • single-stranded serves as template for synthesis of complementary strand

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Why do Eukaryotic DNA molecules have more than one origin of replication, whereas circular DNA only has a single one?

Linear chromosomes of eukaryotic DNA are too large to be replicated quickly enough from a single origin

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Which modes DNA replication are unidirectional or bidirectional, or both?

  • Unidirectional = Rolling-circle

  • Unidirectional or Bidirectional = Theta

  • Bidirectional = Linear Eukaryotic

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Which type of replication requires a break in the nucleotide strand to get started?

Rolling circle replication

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Describe Eukaryotic DNA replication and what it produces:

Replication forks of adjacent replicons run into each other and fuse to yield 2 identical linear molecules

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How does the rate of eukaryotic replication compare to prokaryotes?

20x slower

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What are the 3 main requirements of replication?

  1. A template consisting of single stranded DNA

  2. Raw materials (substrates) to be assembled into a new nucleotide strand

  3. Enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule

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What are the raw materials from which new DNA molecules are synthesized?

deoxyribonucleoside triphosphates (dNTPs)

  • deoxyribose sugar + nucleoside base + 3 phosphate groups

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Where and how are nucleotides added during DNA synthesis?

DNA polymerase adds nucleotides to the free 3’-OH group on the growing strand

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In which direction is DNA synthesized?

5’ to 3’ direction

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How does the phosphodiester bond between the last nucleotide on a strand and the new nucleotide form?

  • 3’-OH group of last nucleotide attacks 5’-phosphate group of incoming dNTP

  • 2 phosphate groups are cleaved from the incoming dNTP

  • phosphodiester bond created between the 2 nucleotides

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Does DNA synthesis occur spontaneously?

No, requires host of enzymes and proteins that function in a coordinated manner

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Why is there a leading strand and laggig strand

because the template strands are anti-parallel but DNA can only be synthesized in the 5’ to 3’ direction

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Leading strand

new strand synthesized in same direction as the movement of the replication fork (direction of unwinding)

  • unwinding → & synthesis →

  • continuous replication

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Lagging strand

new strand synthesized in the opposite direction as the movement of the replication fork (direction of unwinding)

  • unwinding → & ← synthesis

  • discontinuous replication

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Discontinous replication

  • runs out of template as it proceeds away from replication fork

  • synthesis starts again further up template as fork unwinds it

  • short fragments of DNA produced = okazaki fragments

Lagging strand

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Okazaki fragments

short length of newly synthesized DNA produced by discontinuous replication on lagging strand

  • eventually joined together by DNA ligase

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Discontinous replication is a result of which property of DNA?

antiparallel nucleotide strands

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Which enzyme breaks hydrogen bonds at each replication fork?

helicase

  • Unzips

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What is the function of single-stranded binding proteins?

prevent the single stranded DNA template from reforming base pairs after helicase unzipped them

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Which enzyme relieves tension (supercoils) in the helix ahead of the replication forks?

Topoisomerase or Gyrase

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What is the function of DNA polymerase?

adds new nucleotide to 3’ end of growing daughter strand

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Which enzyme joins the okazaki fragments?

DNA ligase

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What is the function of Primase?

Forms RNA primers to initiate replication

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What are the 4 stages in which replication takes place?

  1. Initiation

  2. Unwinding

  3. Elongation

  4. Termination

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What are the key players in the Initiation stage of replication?

  • Initiator proteins

  • Helicase

  • Single-Stranded Binding proteins

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Describe the Initiation stage of replication:

  1. Initiator proteins (DnaA) bind to the oriC (origin of replication)

  2. = short stretch of DNA unwinds

  3. Unwinding allows helicase and single-stranded binding proteins to attach to the single stranded DNA

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What are the key players in the Unwinding stage of replication?

  • Helicase

  • single-stranded binding proteins

  • DNA gyrase

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Describe the Unwinding stage of replication:

  1. DNA helicase binds to lagging strand template at each replication fork and moves 5’ to 3’, breaking hydrogen bonds and moving R. fork

  2. Single-stranded binding proteins stabilize the exposed single -stranded DNA

  3. DNA gyrase relieves strain ahead of replication fork

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How does DNA synthesis begin if DNA polymerases require an existing 3’-OH group to add a new nucleotide to?

Primase synthesizes short stretches of RNA nucleotides (primers), providing a 3’-OH group

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What are the key components of the Elongation stage of replication?

  • Primase

  • DNA polymerase III

  • DNA polymerase I

  • DNA Ligase

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Describe the Elongation stage of replication:

  1. Primase make short stretched of RNA nucleotides.

  2. = Provides free 3’-OH group for DNA polymerase III to add DNA nucleotides to.

  3. DNA polymerase I replaces RNA nucleotides of primer with DNA nucleotides.

  4. Ligase forms phosphodiester bond between 3’-OH and 5’-phosphate groups without adding another nucleotide = joins Okazaki fragments and seals breaks in sugar-phosphate backbone

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Where are primers added during Elongation?

  • 1 primer at 5’ end of newly synthesized leading strand

  • At beginning of each Okazaki fragment on lagging strand

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Why is a nick/break left in the sugar-phosphate backbone after all the nucleotides of new strand are added?

The 3’-OH group of the last nucleotide replaced by DNA polymerase I is not attached to the 5’ phosphate group of the 1st nucleotide added by DNA polymerase III

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When does DNA know when to Terminate (end) replication?

  • some terminate when 2 replication forks meet

  • some require specific termination sequences (Ter sites) to block further replication

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Tus-Ter complex

Ter site and termination protein complex that blocks movement of helicase = stalling replication fork

  • blocks replication fork from moving in one direction but not the other

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What is the error rate of DNA replication?

less than 1 mistake per billion nucleotides

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How are errors in DNA polymerase III base-pairing corrected?

corrected immediately using DNA polymerase’s 3’ to 5’ exonuclease activity and repair

  • backwards removal and repair = proofreading

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How are any mistakes that are present after DNA replication is completed rectified? How are the old and new strands identified?

Mismatch repair enzymes correct any error that remains after DNA replication is completed

  • excise incorrectly paired nucleotides from new strand and use original strand as template to replace them

  • In bacteria, original DNA is methylated, so repair enzymes know which strand to fix

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Why is gene sequence erosion not an issue in the circular DNA of bacteria?

there is always a 3’-OH end, no need for the RNA primers to be removed and replaced

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Why does gene sequence erosion occur in linear chromosomes?

  • end of chromosome has no 3’-OH group preceding the primer

  • terminal primer removed = cannot be replaced by DNA nucleotide

  • results in gap at end of chromosome

  • = chromosome progressively shorter with each round of replication

  • = gene sequence eventually eroded and cell dies

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How do telomeres relate to gene erosion?

  • heterochromatic regions of many short repeated sequences at ends of linear chromosomes

  • postpone erosion of vital gene sequences

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What would be the result if an organism’s telomerase were mutated and nonfunctional?

chromosomes would shorten with each new generation

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How does telomere shortening relate to aging?

telomere shortening may contribute to aging

  • most somatic cells have a pre-programmed life span with limits on how many times they can divide before death (apoptosis)

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How do germ line (produce gametes) cells, single cell organisms, proliferative cell types prevent telomere shortening?

using telomerase

  • enzyme that restores telomere length

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Telomerase

enzyme that restores telomere length

  • composed of protein and RNA components complementary to G-rich strand that serves as template for DNA synthesis

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Describe how telomerase restores telomere length:

  1. Telomere has protruding end with a G-rich repeated sequence

  2. RNA part of telomerase complementary to G-rich and pairs w it = template for synthesis of copies of the repeats

  3. Nucleotides are added to 3’ end of G-rich strand