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Lecture 12

BIOB11H3 Lecture Notes - Lecture 12: Chromosome, Polymerization, Dna Polymerase Iii Holoenzyme


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs
Lecture
12

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BIOB11: Molecular Aspects
56
Lecture 12: DNA Replication I: General Aspects & Enzymology
-Watson & Crick drew double stranded helix that was anti-parallel and each strand was a template
for replication of a new strand
3 Possible Replication Mechanisms:
*each strand serves as a template for the formation of the complementary strand
*parental strand is copied into daughter strand
1.) Semiconservative
-Parental/daughter strand hybrids
-Energy & time efficient; less prone to errors
2.) Conservative
-Parental strands together
-Original strands must rejoin each other
3.) Dispersive
-“Random” parental/daughter strands
-Requires numerous fragmentation & rejoining events
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BIOB11: Molecular Aspects
57
Meselson & Stahl Experiment
-used E. Coli to investigate the mechanism of replication
-Employed a density label: heavy Nitrogen (N15) (supplied as 15NH4Cl)
-Method:
1.) Grow cells for many generations in N15 medium
>uniformly label DNA with N15
2.) Wash cells and switch to normal N14 medium
>upon replication, DNA would now have some N15 & N14
>Thus, density of DNA would differ
3.) After different time points, purified DNA separated by centrifugation and the N14/N15 ratios are
determined
>Equilibrium Density Gradient Sedimentation
-Utilizes a dense medium (ex.: 6M
CsCl) and particles sediment until
they reach their bouyant density
(equilibrium)
-subject to extended centrifugation
Possible Results:
*Light: N14/N14
*Hybrid: N14/N15
*Heavy: N15/N15
Actual Results:
Conclusion: Mechanism is Semiconservative
-since half are N15/N14 and half are N14/N14
-didn’t align w/ conservative, otherwise all N15
-didn’t align w/ dispersive, otherwise all N14
Proof of Semiconservative Mechanism in Eukaryotes
-by J.H. Taylor
-cells grew in Thymidine(T) medium then moved into
Bromodeoxyuridine(BrdU) to replicate twice
>one chromatid of each chromosome contained T
I
II
Generations
III
Parental
Transfer to 14N
Light 14N DNA
Hybrid 14N15N DNA
Heavy 15N DNA
I
II
III
I
II
III
(a)
Semiconservative
Conservative
Dispersive
Heavy
Hybrid
Light
Increasing
Density
N14/N14
N14/N15
N15/N15
After one round of replication all
molecules sediment at the hybrid
(N14/N15) density
After two rounds of replication, half
of the molecules are hybrid and
half are light (N14/N14)
Chromosome contains
only thymidine
Replicates
in BrdU
Chromosome
Both chromatids contain one strand with BrdU and
one strand with thymidine
Continued replication
in BrdU-containing
medium
One chromatid of each chromosome
contains thymidine
(a)
Chromatids
DNA strand
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