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Chapter 7 DNA: Structure and Replication p.

251-279
Friday, May 8, 2015
8:30 PM

-Watson and Crick concluded that DNA is a double helix composed of 2 strands of linked
nucleotide bases that wind around each other:
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Information for making an organism is encoded in the sequence of the nucleotide bases
composing the 2 DNA strands of the helix
Because of the rules of base complementarity the sequence of one strand dictates the
sequence of the other strand

7.1 DNA: The Genetic Material

Discovery of Transformation (Griffith Experiment)
Killed polysaccharides, lipids, proteins, RNA, and mice died, but when DNA was killed
mice lived. (They were injected with a deadly virus)

Hershey Experiment
Used radioisotopes for proteins and DNA separately and saw that children had only
glowing DNA

7.2 DNA Structure Before Watson and Crick
Phosphate + Deoxyribose Sugar (One hydroxyl group) + Nitrogeneous base


Purine: a double ring structure that Adenine and Guanine have.
Pyrimidine: a single ring structure that Cytosine and Thymine have
Nucleotide = one phosphate group + one deoxyribose sugar + one of four nitrogenous
bases

Chargaff's Rules of Base Composition

Amount of (T+C) = (A+G)
Amount of (A+T) NOT = (C+G)

The Double Helix



Base pairs are flat planar structures and are stacked on top of one another to exclude
water molecules for stability
Two distinct sizes of grooves: Major and Minor Grooves
Purine bases always pair with Pyrimidine bases so same radius
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Note: C-G pairs have 3 hydrogen bonds and A-T has 2 hydrogen bpnds

7.3 Semiconservative Replication
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Semiconservative Replication: the double helix of each daughter DNA molecule contains
one strand from the original DNA and one newly synthesized strand
Conservative Replication: the parent DNA molecule is conserved, and a single daughter
double helix is produced consisting of two newly synthesized strands
Dispersive Replication: the daughter molecules consist of strands each containing segment
of both parental DNA and newly synthesized DNA

Meselson-Stahl Experiment

Provided evidence for Semiconservative Replication by placing high density DNA in
Low density DNA and obseving in the F1 generation an intermediate density DNA and in
the F2 generation an intermediate density DNA and low density DNA.

The Replication Fork

An Autodiograph of bacterial DNA showed two progressing replication forks.

DNA Polymerase


How the bases are brought to the double-helix template
Substrates are the triphosphate forms of the deoxyribonucleotides: dATP, dGTP, dCTP,
Dttp
Addition of each base to the growing strand is accompanied by the removal of two of the
three phosphates in the form of phyrophosphate (PPi)
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Note: The energy produced by cleaving this high-energy bond and the subsequent
hydrolysis of pyrophosphate to two inorganic phosphate molecules helps drive the
endergonic process of building a DNA polymer.

7.4 Overview of DNA Replication

As DNA polymerase pol III moves forward, the double helix is continuously unwinding
ahead of the enzyme to expose further lengths of single DNA strands that will act as
templates.
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Acts at the replication fork, the zone where the double helix is unwinding
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Proceeds in a smooth and continuous manner at the 3' growing strip of only one
strand called the leading strand.


Synthesis on the other strand which the 5' to 3' direction is moving away from the
replication fork is in short segments called Okazaki Fragments
DNA polymerase can only extend a chain but not start one so it is initiated by a primer, or
short chain of nucleotides.
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These primers are synthesized by a set of proteins called a primosome, of which a
central component is an enzyme called primase, a type of RNA polymerase.
On every leading strand only one initial primer is needed but on the

lagging strand every Okazaki fragment needs its own primer.

Then is extended as a DNA chain by DNA pol III
DNA polymerase, pol I, removes the RNA primer with its 5' to 3'

polymerase exonuclease activity and fills in the gaps with its 5' to 3' polymerase
activity.
DNA ligase joins the 3' end of the gap filling DNA to the 5' end of the downstream
Okazaki fragment
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That is why it is called the lagging strand
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Does this by catalyzing the formation of a phosphodiester bond between the 5'phosphate end of one fragment and the adjacent 3'OH group of another fragment
This process is very accurate one error per 10^10 nucleotides because DNA pol I and pol
III both have 3' to 5' exonuclease activity which serves as a proofreading function.
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Addition of an incorrect base occurs because of tautomerization. Bases come in
different forms (different bonds and positionings) ,but in DNA usually in keto form.
However, sometimes might shift to imino and enol forms which might pair with the
wrong base
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This is detected and removed by the 3' to 5' exonuclease activity
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That is why RNA primer is removed because it has higher rates of this

7.5 The Replisome: A Remarkable Replication Machine


DNA replication is very fast
DNA polymerase is part of a large "nucleoprotein" complex that coordinates the activities
at the replication fork called the Replisome.
DNA pol III is part of a much larger complex called the pol III holoenzyme, which
consists of two catalytic core and many accessory proteins.
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Some of the accessory proteins form a conection that bridges the two catalytic
cores, thus coordinating the synthesis of the leading and lagging strands.
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The accessory protein Beta clamp encircles the DNA like a donut and keeps pol
III attached to the DNA molecule
So It doesn’t fall off after 10 nucleotides called a Distributive Enzyme and

instead stays for thousands called a Processive Enzyme.

Unwinding the Double Helix

Two classes of proteins that open the helix and prevent overwinding: they are Helicase
and Topoisomerases, respectively.


The unwound DNA is stabilized by single-strand-binding (SSB) proteins, which bind to
single-stranded DNA and prevent the duplex from reforming
Circular DNA can be twisted and coiled, much like the extra coils that can be introduced
into a rubber band ------- extra twisting at other regions called supercoils form to release the
strain of the extra twisting. This supercoiling can be created or relaxed by enzymes termed
topoisomerases

Assembling the Replisome: Replication Initiation for Ecoli

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Occurs at precise sites on the chromosome called origins
The binding of a protein called DnaA to a specific 13 base pair sequence that is repeated
five times in oriC (origin)
In response to the binding of DnaA, the origin is unwound at a cluster of A and T
nucleotides (because it is easier)
Additional DnaA proteins bind to the unwound DNA, then two helicases bind and start
moving in the 5' to 3' direction.
Primase and DNA pol III are recruited and begin DNA synthesis

7.6 Replication in Eukaryotic Organisms

Because Eukaryotes are more complex their replisome has more components

The Eukaryotic Replisome

Eukaryotic Chromosomes exist in the nucleus as chromatin
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The basic unit of a chromosome is the nucleosome, which consists of DNA
wrapped around histone proteins
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To disassemble this and create new histones, CAF-1 protein takes histones and
arrive at replication fork and latch on to the eukaryotic version of the beta clamp, the
proliferating cell nuclear antigen (PCNA)

Eukaryotic Origins of Replication

Thus in Eukaryotes, replication proceeds in both directions from multiple points of origin

DNA Replication and the Yeast Cell


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DNA synthesis takes place in the S phase of Eukaryotic cell cycles
In yeast three proteins are required to begin the assembly of the replisome
ORC (origin recognition complex) at the origin recruits two other proteins Cdc6 and Cdt1.
Called the MCM complex
They bring the helicase and other components of the Replisome
Replication is linked to the cell cycle to the availability of Cdc6 and Cdt1 and occur before
the S phase

Replication Origins in higher Eukaryotes

Origins much bigger

7.7 Telomeres and Telomerase: Replication Termination



Inherent problem in replicating the two ends of linear DNA molecule, the regions called
telomeres
Lagging strand is missing a sequence at the end of that strand.
Solution: addition of multiple copies of a simple noncoding sequence to the DNA at the
chromosome tips. So every time a chromosome is duplicated, it is shortened and only
repeating sequences are lost and the lost repeats are then added back to the chromosome
ends
At the 3' end called overhang
Telomerase adds the short repeats to the 3' ends of DNA
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Carries a small RNA molecule which acts as a template for the telomeric repeat
unit
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The telomerase moves extending the 3' end creating a primer and the DNA
polymerase fills the gap and then the primer is removed and the ligase seals the gap.