You are on page 1of 36

L8

DNA
STRUCTURE

is key to understanding:

(1) replication;

(2) inheritance;

(3) allele diversity;

(4)mutation; and

(5) protein

expression and variation

1850-1950: In historical order, major landmarks in genetics





(1)Mendel: genes hereditary particles, gene alleles

correlated with character states,

tested in breeding designs and the logic extended to pedigrees,
other allelic and genetic interactions, complementation, and
mutant screens.



(2) Genes are on certain chromosome locations (T.H. Morgan
and A. Sturtevant 1910-1920), and crossover involves breakage
and rejoining (H. Creighton and B. Mclintock 1931).



(3) Strain types (genes) can be transformed into different types by
the presence of other dead strains (Griffiths 1928).



(4) One gene related to one enzyme in pathway analyses - G. W.
Beadle and E. L. Tatham (1941) - then one polypeptide .


DNA : discovery of the


genetic material.

In 1928 Frederick Griffiths demonstrated the existence


of a transforming principle that changed the form
and function of a bacterial strain type of Streptococcus
pneumoniae into another type.



A non-virulent strain that formed rough-looking colonies (R)

was transformed, to a virulent (geno)type associated with
smooth (S) colonies. However, this could only be attributed to a
principle

3

Transformation principle: unexpected results and the


empirical value of experimental controls in an in vivo study.

Heat 60 oC =transformation; no transformation > 80 oC ,
freezing & thawing or old cultures

5

In 1931, R. Sia and M. Dawson performed the same
experiment in vitro, in liquid culture, showing .

In 1931, R. Sia and M. Dawson performed the same


experiment in vitro, in liquid culture, showing .



a) Better controls were necessary.

b) The host was not required for bacterial
transformation.

c) In vitro is a preferred technique for showing bacterial
uptake is not mediated by some other process.

d) Experiments should be replicated to be believable.

Beginnings of molecular genetics



In 1944 the laboratory of Oswald Avery, Colin MacLeod and
Maclyn McCarthy identified the biochemical class of the
transforming principle, following Sia and Dawsons
experiment.



First they developed an assay measuring the conditions
wherein R cells of an optimal strain could be reliably
transformed by a volume of a cell free extract of S cells.



Using selective elimination of the major biochemical
categories, They showed that only DNA deoxyribonucleic
acid, was capable of replicating the transforming principal
effect.

6



Surprisingly, it was not in a protein, not a carbohydrate, not a
lipid, nor RNA.



(1) Standard biochemical purification indicated that DNA is
the only class of molecules that would transform R to S
(same chemical composition, density, absorbed UV like DNA).

(2) A second experiment used enzymes that degrade DNA
(ases), RNA(ases) and proteins (proteases), destroys the
T effect.

Avery, McLeod and
McCarthy 1944

In 1952 Alfred Hershey and Martha Chase confirmed that



DNA was the genetic material, at least in virus.



At that time the method of phage replication was not known,
but it was known that the T2 phage was approximately 50%
protein, 50% DNA.



They used radioactive isotopes of phosphorus and sulphur

to label components of T2 phage.



32P labelled the DNA, 35S labelled the protein.



Only the 32P , i.e. DNA, of T2 appeared to enter the bacterial
cell mediating the infection and replication of the phage.

Importantly, after the cells lysed and the progeny phage emerged
many were labelled with 32P.

8

No transmission of
S35

Transmission of P32

The Chemical Foundations of Watson & Cricks


discovery:



Why Griffiths, Sia & Dawson, Avery, McLeod and
McCartys results were so surprising is that from a
chemical point of view, DNA comprises only:




1 Phosphate group,

1 sugar (Deoxyribose) and

4 types of nucleotides.



Somehow this simple molecule class explains:

(1) faithful replication (2) information -the enormous
diversity of inheritance, and (3) mutation. to begin
with.

10

DNA STRUCTURE 1: Four nitrogenous bases, sugar and


phosphate

Four nucleotides of 2 types


11

The % A and C or G and T in the DNA from different species was


different.



But the % A always is (approximately) equal to the % T and the % G is
always (approximately) equal to the % C.

Chargaffs rule:

A ~ T and G ~ C, but,

12

1850-1950: DNA is the Genetic Material, but how does it work?





(1)Mendel: genes hereditary particles, gene allele expression

correlated with visible character states





tested in monohybrid breeding designs.

(2) Genes are on certain ordered, chromosome locations (T.H.
Morgan and A. Sturtevant 1910-1920), and crossover involves
breakage (H. Creighton and B. Mclintock 1931)

(3) Genes can be transformed by other alleles (Griffiths 1928).

(4) One gene one enzyme, then one polypeptide G. W. Beadle
and E. L. Tatham (1941) . First protein sequence (1951)

(5) Transforming Principal is in bacterial DNA (Avery and
McLeod 1944).

(6) Hersey Chase DNA is transmitted between viral generations
(1952).

(7) Chargaffs rules


(1) The Double Helix



covalent bond
(outside)

13

The resolution of
the B form :

Each turn of the
helix is 10 base
pairs, separated
by 3.4 A

James Watson and Francis Crick, in 1953, worked with the X-ray
diffraction data of Rosalind Franklin and Maurice Wilkins to propose
the double helix structure of DNA. Read J. Watson The Double Helix.

J. Watson on R. Franklin The real problem was Rosy.


There was no denying she had a good brain. If she could
only keep her emotions under controlThe thought
could not be avoided that the best home for a feminist
was in another persons lab. pp 15 The Double Helix

Chargaff I told them all I knew. If they had heard
before about the pairing rules, they concealed it. But as
they did not seem to know much about anything, I was not
unduly surprised. I mentioned our early attempts to
explain the complementarity relationships by the
assumption that, in the nucleic acid chain, adenylic
was always next to thymidylic acid and cytidylic next
to guanylic acid...I believe that the double-stranded
model of DNA came about as a consequence of our
conversation.

DNA density

2 polynucleotide
Francis Crick (right) and James Watson (left)
and their structural model of B DNA,

Chains.

34A/turn

20A
diameter

3.4A
separation
between
nucleotide
s

10 nucleotides per complete 1 (360)

helical turn

10A = 1nm

14

The Problem Watson and Crick took on

15

What are the requirements for genetic material explaining the inheritance of molecules,
physiology, morphology, behaviour etc.
A code that is: (1) stable (2) replicable (3) expressible (4) can evolve
In April 1953, Watson and Crick proposed a structural model in Nature:
We propose
the double helix, (stable structure)
complementary base pairing (expressible) and
a mechanism for DNA replication by unwinding and separating
Followed by a scale model (1954 Proc. R. Soc. (A): 80 - 96) they also proposed a mechanism
for mutation (can evolve)
Several years later, Crick et al. (1961) provided evidence that the code involved triplet base
sequences.

DNA Stability



(1)A large number of hydrogen bonds joining
strands.

(2)Each strand has a chain of covalently bonded,
phosphate-nucleoside units (backbone).

(3)A-T, C-G pairs (purine -pyrimidine) same
diameter along the length, no bulges in the helix.

(4)Charged groups face outward into waterinteract strongly.

(5)Helical structure means the inner H -bonded
nucleotides are protected from interacting with
water

16

2-dimensional view of DNA





Note:

!C=G and A=T nucleotide pairing

differs in the number of H bonds

!Deoxyribose-phosphodiester

backbone.

!directionality of each strand (5 to 3).

The two strands are antiparallel,

one going 3 to 5, the other 5 to 3

17

Hydrogen bonding between nucleotide pairs.



Individual hydrogen bonds are much weaker than covalent
bonds, but 3 bonds >2, and there are many of them.

18

3-5 covalent
phosphodiester bond
between two
nucleosides

(deoxyribose + base)

Forms the backbone
of each strand

19

Complementary
structure
implies different
strand
directions

20

Covalent bonds are strong. Each strand of the double



helix is composed of atoms (C,N,O,P, H ) bound by strong
(charged) covalent bonds in the phosphodiester backbone.
Because they are negatively charged they interact strongly
with water (hydrophyllic).



Hydrogen bonds are relatively weak, hydrophobic bonds.
The two strands of the double helix are held together by
hydrogen bonds. But, because (1) there are many hydrogen
bonds between the two strands,(2) the bases are stacked (3)
purine (large) are paired with pyrimidine (small) rings and (4)
water is on the outside, DNA structure is stable.



If DNA is denatured by high temperature or low salt
concentration, the two strands unfold (hydrogen bonds are
broken) , but each strand stays intact.

21




DNA is coiled in a right handed helix B


DNA

22

The major (22A across) and minor grooves (12 A) are


important for proteins that regulate gene expression.

23

There was one other important


24

level of organization in functional
DNA, they are supercoiled no free
ends. Linear chromosomes are
anchored to a protein scaffold.



Almost all organisms have
negatively supercoiled DNA, and
many biological functions can only
be carried out with negatively
supercoiled DNA (replication ,
recombination gene expression and Twist -#helical turns.

regulation) in both bacteria and Writhe -# times double helix crosses
itself

eukaryotes.



Supercoiling produces tension in
22

DNA structure, which is probably
important in replication

The structure of DNA presents a mechanism to account for



two key functions of genetic material.



1. Complex information storage.



The vast variety of A, T, C and G sequence along a

DNA strand can contain complex information.



2. Replication.



The double stranded complementary base structure
facilitates rapid and accurate SEMI CONSERVATIVE
duplication of the DNA.


25

26

Three possible

models for

DNA replication.





The first test of
Watson & Cricks
semiconservative
model was
reported in 1958!

1958: Mathew Meselson and Franklin Stahl tested


three possible models of replication.

(1) They grew E. coli in the presence of a heavy isotope of
nitrogen, 15N. This produced DNA of high density.



(2) They followed this with growth in normal 14N for one cycle

and

(3) A second cycle of replication in 14N.



DNA density was measured by centrifuging in an ultracentrifuge

in a cesium chloride gradient.

27

28

29

30

The three models



of replication give

three different

predictions of DNA

density after one and

two rounds of

replication.

Messelson and Stahls



data showed that

DNA is replicated

semi-conservatively.

31

The take-home message here is that the structure of DNA, is


stable, replicable, expressible, mutable and determines the
protein code , or structure and function are inseparable

What are the requirements for genetic material explaining the inheritance of molecules,

physiology, morphology,
behaviour
etc.

A code
that is:
(1)
stable
(2)
replicable
(3)
expressible
(4)
can evolve

In April 1953, Watson and Crick proposed a structural model in
Nature
:



We propose


the double helix,
(stable structure)


complementary base pairing (
expressible
) and


a mechanism for DNA
replication
by unwinding and separating


Followed by
a scale model
(
1954 Proc.

R.

Soc. (A): 80 - 96
) they also proposed a mechanism

for mutation (
can evolve
)

Several years later,
Crick et al. (1961
) provided evidence that
the
code involved triplet base

sequences
.