DNA Replication 2 Flashcards
1
Q
Three Models of DNA Replication
A
- Semi-conservative model: Parental strand and newly synthesized strand remain associated. Each newly formed DNA molecule contains one parental strand and one newly synthesized strand
- Conservative model: Two parental strands and two newly synthesized strands reassociated with each other. One molecule consist of two parental strands, while other consist of two newly synthesized strands
- Random dispersive model: parental DNA molecule breaks into fragments, each fragment serves as template for synthesis of portion of new DNA strand. Newly formed DNA molecules contain mixture of old and new DNA segments
2
Q
Meselson and Stahl Experiment
A
- Cells grown in N15 medium: Nitrogen molecules in nitrogenous bases in DNA replaced with N15
- Cells transferred to N14 medium: All DNA synthesized after medium switch contain N14, and are less dense
- DNA subjected to cesium chloride gradient: collected DNA samples were centrifuged and separated based on density. More dense molecules migrate farther down tube
- Visualization of DNA: Intercalating agent added for visualization, followed by UV light exposure. Revealed positions of different DNA molecules within gradient
3
Q
Predictions
A
- Conservative model: Show distinct bands after one round of replication. Less dense band represent DNA molecule with two newly synthesized strand made of N14. Denser band represent a DNA molecule composed of two N15 parental strand
- Semi conservative model: Single band after one round of replication representing two DNA molecule of same density. Composed of one newly synthesized N14 strand, and one parental N15 strand
- Random dispersive model: Single band after one round of replication. DNA molecule composed of 50% parental and 50% newly synthesized, with each individual strand containing both segments
4
Q
Distinguishing between semi-conservative and random dispersive
A
- Compare at second round of replication
- Random dispersive would produce single band of same density. Each DNA molecule composed of 25% N15 DNA, 75% N14 DNA
- Semi conservative would produce two bands at different densities. Denser band represent two DNA molecule composed of N15 template strand and newly synthesized N14 strand. Less dense band represent N14 used as template and another newly synthesized N14
- Results show DNA replication is semi-conservative
5
Q
Huberman and Riggs on Unidirectionality or Bidirectionality
A
- Accumulate cells in S phase: Mammalian cells exposed to 5-fluorodeoxyuridin for 12 hours. Inhibits thymine synthesis and DNA replication. Arrest at S phase -> poised to start with addition of thymine
- Pulse: Cells exposed to radioactively labeled dTTP -> allowed cells to enter S phase. dTTP incorporated to start of replication bubble
- Chase: Excess normal dTTP added to cells. Likelihood of radiolabeled dTTP added is decreased. Dilution effect: concentration of radiolabeled dTTP highest near starting point and gradually decreased in direction of replication
- Fibre autoradiography: used to visualize incorporation of radiolabeled dTTP
6
Q
Results
A
- Bi-directional replication: Two replication forks and bubble move and replicate it opposite directions
- Multiple origins of replication
7
Q
Okazaki on how template in wrong direction could be replicated
A
- Continuous: Replication proceed continuously on both strands
- Semi-discontinuous: Wrong direction synthesize in pieces, and right direction synthesize continuously
- Discontinuous: Replication discontinuous on both strands
- Pieces: template strand cleaved into pieces during DNA replication and replication discontinuous on both strands
8
Q
Methods
A
- Start reaction: Replicating E.coli cells pulsed with radiolabeled dTTP
- Stop reaction: Reaction stopped at different time points by pouring culture over ice with cyanide
- Ultracentrifugation in sucrose alkaline gradient: Allowed for separation of fragments by weight, heavier fragments at bottom and lighter at top. Alkaline buffer denatures DNA
- Fractionation: Larger fragments exited first and collected in new fractionation tube. Smaller fragment exited last and collected in separate fractionation tube
- Measure: Radioactivity of each fractionation tube measured and plotted on a graph. Each data point represents amount of DNA of a specific size that was synthesized at a specific time point
9
Q
Results
A
- Early time point: From beginning, DNA replicating as smaller and larger species indicated by presence of 2 peaks
- Middle time point: Concentration of short DNA fragment increasing. Concentration of large DNA fragment also increasing and getting longer.
- Late time point: Concentration of short DNA fragment decreased due to ligation of short okazaki fragments into larger DNA fragments
- DNA replicates semi-continuously
10
Q
DNA helices
A
- Answers how DNA is unwound into single stranded DNA during replication
11
Q
Prokaryotic helicases
A
- Toroid homo-hexamer (six polypeptides)
- ssDNA passes through central cavity
- Move on lagging strand 5’ - 3’
- Positive charge of central cavity to neutralize DNA backbone
12
Q
Eukaryotic helicases
A
- Toroid hetero-heamer (each subunit has same ATPase domain)
- Bind both ds and ssDNA
- Move on leading strand 3’-5’
- Positive charge of central cavity to neutralize DNA backbone
13
Q
Single stranded DNA binding protein
A
- ssDNA can reanneal, form secondary structures, or undergo nucleolytic cleavage
- Eukaryotic binding protein known as Replication protein A (made of RPA70, RPA32, and RPA14)
- Three binding domains total (two in RPA70 and one in RPA32)
14
Q
DNA Primase
A
- Provides 3’-OH ends to initiate replication
- pola primase complex responsible for de novo synthesis of primer with both DNA and RNA subunits
- Composed of four subunits: primase component (P58 and P48) synthesize RNA segment of primer. poly-a component (P180 and P86) synthesizes DNA segment
- Primase component synthesizes 11-14 nucleotide RNA primer -> poly-a component extends by 10-20 nucleotides
- Has low fidelity
15
Q
Topoisomerase
A
- Opening of DNA causes torsional strain to helix causing supercoiling
- Topoisomerases break phosphodiester bond downstream of replisome to relieve strain
- Type 1: single strand break
- Type 2: double strand break
16
Q
Sliding Clamp
A
- Ring shaped protein complex encircling DNA double helix
- Anchors DNA polymerase to keep from dissociating
- Sliding clamp called proliferating cell nuclear antigen (PCNA) in eukaryotes
17
Q
Structure and function of PCNA
A
- Resemblance to DNA helicase with six six-fold axis of symmetry and central channel
- Homotrimer with three identical subunits, lacks ATPase activity
- only DNA pol epsilon and DNA pol delta associate with sliding clamp (responsible for leading and lagging strand synthesis)
- DNA pol a does not bind to PCNA because of low fidelity
18
Q
Assay for sliding clamp activity
A
- absence of PCNA resulted in synthesis of small DNA fragments -> DNA polymerase frequently started and stopped synthesis
- In presence of PCNA, large DNA fragments occurred
19
Q
Loading clamp onto DNA
A
- Replication factor C (RFC) associates with PCNA through RFC subunits 1, 3, 4
- RFC binds ATP, additional contact to PCNA through RFC subunit 2, 5 -> opens PCNA donut
- RFC and open clamp binds to primer/template junction
- DNA binding promote ATP hydrolysis by RFC causing PCNA ring to close around double-stranded DNA -> dissociation of RFC from PCNA
- DNA synthesis begins
20
Q
Replisome
A
- Answers how synthesis of two strands is coordinated
- Replisome comprised of DNA polymerase, helicase, primase, sliding clamp, clamp loader, and single strand binding proteins.
- Moves along dsDNA, synthesizing both leading and lagging strands simultaneously
