ch 9 chemistry of gene

8/8/2019 Ch 9 Chemistry of Gene

1/50

Chemistry of Gene

Chapter 9 of TB


2/50

In 1953 Watson and Crick proposed the model for the double helix structure

of DNA.


3/50

Evidence for DNA as the Genetic material:

Transformation:

In 1928, F. Griffith reported that heat killed bacteria of one typecould transform living bacteria of a different type.


4/50

In 1944, Oswald Avery along with Mac Leod and McCarty reported the nature

of the transforming substance.

Chemical analysis showed that the extract of the transforming substance was

made up of DNA, RNA, proteins and lipids.

They worked in vitro and ruled out the possibility of lipids, RNA and protein

by using lipases, Ribonuclease and trypsin and chymotrypsin.

Enzymes that destroyed the DNA also destroyed the transforming principle.

Thus, confirming the transforming principle to be DNA.

This study provided the first experimental evidence that DNA was the genetic

material.


5/50


6/50


7/50

RNA as Genetic Material:

In some viruses, RNA is the genetic material.

Eg: Tobacco mosaic virus, contains only RNA and proteins, Influenza

virus, Hepatitis C virus


8/50

Chemistry of Nucleic Acids:

Nucleotides: Sugar, phosphates and a

nitrogenous base.

Nucleoside: sugar and a nitrogenous base


9/50


10/50


11/50


12/50


13/50

Chargaff rule states that the number of purines is always equal to the

number of pyrimidines in a given DNA. The relationship is that the number

of adenine residue equals the number of thymine, and the number of

guanines equals the number of cytosine that is A = T and G = C. Thus we cansay A + G = C + T.

X-ray diffraction studies X-ray diffraction studies by M. Wilkins and R.

Franklin suggested that DNA could be a helix with two regular periodicities

of 3.4 A and 34 A along the axis of the molecules.


14/50


15/50

4. The nitrogenous bases are stacked towards the inside of the helix. The

experimental evidence also indicated that the sugar-phosphate backbone of

the molecules is on the outside, with the bases inside the helix.

5. Bases of the two polynucleotides interact by hydrogen bonding. This is

the explanation of Char gaffs base pairs.An adenine residue in one of the

polynucleotides is always adjacent to a thymine in the other strand;

similarly guanine is always adjacent to cytosine. These two pairs of bases,

and no other combinations are able to form hydrogen bonds between each

other. These hydrogen bonds are the only attractive forces between thetwo polynucleotides of the double helix and serve to hold the structure

together.


16/50


17/50

Double helix structure of DNA


18/50


19/50

Alternative forms of DNA:

The form of DNA described so far is the B DNA.

It is right handed helix ie it turns in a clockwise manner when viewed down

its axis.

The bases are stacked almost exactly perpendicular to the main axis, with

about 10 base pairs per turn (34 A).

If the water content of this form of DNA increases to 75%, the A form of DNA

(A DNA) occurs.

In the A DNA, there are more base pairs per turn. However, this form of DNA

is a minor variant.

In 1979, a left handed helical DNA was discovered, termed Z DNA.

The backbone of this DNA formed a zigzag structure.

Z DNA looks like B DNA with each base rotated 180 degrees, resulting in a

zigzag , left handed structure.


20/50

The Z DNA requires high salt concentration to become stable.

However, it can be stabilized in physiologically normal conditions if methylgroups are added to the cytosines.

Z DNA is thought to be involved regulating gene expression in eukaryotes.


21/50


22/50

DNA Replication:

There are three methods of DNA replicationa. Semiconservative replication: each strand of the DNA is conserved in

the daughter cell. Every daughter molecule has an intact template

strand and a newly replicated strand.

b. Conservative replication: the whole original double helix acts as a

template for a new one. One daughter molecule would consist of the

original parental DNA, and the other daughter would be totally new

DNA.

c. Dispersive replication: some parts of the original double helix are

conserved and some parts are not. Daughter molecules would consist of

part template and part newly synthesized DNA.


23/50

Meselson and Stahl Experiment:


24/50

Autoradiographic demonstration of DNA replication:

I

n 1963, J Crains used autoradiography tov

erify the semiconserv

ativ

emethod of DNA replication.

In this technique photographic film is exposed by radioactive atoms.

The visible silver grains on the film can be counted to provide an estimate of

the quantity of radioactive material present.

Crains grew E.coliin a medium containing radioactive thymine (in tritium3H). He extracted the DNA from the bacterial cells and placed it on the

photographic emulsion ( silver chloride) for a period of time. He developed

the emulsion to produce autoradiographs, which were examined under

electron microscope.

Interpretations of the autoradiographs:

1. E.coliDNA is a circle

2. DNA is replicated while maintaining the integrity of the circle Theta

structure is seen.

3. Replication of DNA seems to occur at one or two moving Y-junctions in

the circle, suggesting the semiconservative mode of replication.


25/50


26/50


27/50

In eukaryotes, the DNA molecules are larger than in prokaryotes and are

not circular.

There are multiple sites for the initiation of replication and hence each

chromosome is composed of many replicating units or replicons.

Stretches ofDNA.

Replication bubbles: formation of bubbles in eukaryotic DNA

because of multiple DNA synthesis sites of origin


28/50

Enzymology:

There are three major enzymes that polymerize nucleotides into a growing

strand of DNA in E.coli.

These three enzymes are DNA polymerase I, II and III.

DNA polymerase I primarily utilized in filling in small DNA segments during

replication and repair processes.

DNA polymerase II

serves as an alternative repair polymerase, it can also

replicate DNA if the template is damaged.

DNA polymerase III primary polymerase during normal DNA replication.

All the known polymerases add nucleotides in only the 5 3direction. Ie

the polymerase requires a free OH group, to which the 5 PO4 of the newnucleotides forms a bond, catalyzed by the polymerase.

All the three polymerases also possess the property ofexonuclease activity

(remove the nucleotides), necessary for the proof reading function.


29/50

Continuous and Discontinuous DNA Replication:

Continuous replication is possible on the 3 5 template strand, whichbegins with the necessary 3OH primer.

Primeris a double stranded DNA or a DNA-RNA hybrid continuing as single

stranded DNA template.

The strand being synthesized has a 3OH available.


30/50


31/50


32/50

Discontinuous replication takes place on the complementary strand, where

it occurs in short segments, moving backward, away from the Y-junction.

These short segments are called Okazakifragments.

The strand synthesized continuously is referred to as the Leading strand.

The strand synthesized discontinuously is referred to as the Lagging strand.

Once initiated, continuously DNA replication can proceed indefinitely. DNApolymerase III on the leading strand has high processivity, ie: once it

attaches, it doesnt release until the entire strand is replicated.

Discontinuous replication however requires the repetition of four steps:

primer synthesis, elongation, primer removal with gap filling and ligation.


33/50


34/50

Primer synthesis and elongation-

To synthesize Okazaki fragments, a primer must be created de novo.

DNA polymerase cannot create the primer.

Primase, a RNA polymerase creates the primer, 10-12 nucleotides at the site

of Okazaki fragment initiation.

Short RNA primers are formed that provide a free 3 OH group that DNA

polymerase needs in order to synthesize the Okazaki fragments.

DNA polymerase III continues until it reaches the primer of the previously

synthesized Okazaki fragment. At this point it stops and releases from the

DNA.


35/50


36/50

Primer removalwithgapfilling

DNA polymerase I is the enzyme involved in Primer removal with gap filling.

It acts as apolymerase when it adds the nucleotides and an exonuclease

when it removes nucleotides.

It removes the RNA primer (exonuclease activity) and replaces it with DNA

nucleotides (polymerase activity).

After completion of the exonuclease and polymerase activity, the Okazaki

fragments are removed and only the phosphodiester bond formation

remains.


37/50


38/50

Ligation

DNA polymerase I cannot make the Phosphodiester bond to join the two

Okazaki fragments.

An enzyme, DNALigase, completes the task by making the final

phosphodiester bond in an energy requiring reaction.


39/50


40/50

Origin of replication :

Each replicon has a region where DNA replication initiates.

In E.colithis region is referred to as the genetic locus oriCand is about 245

bps long and is recognized by the initiator proteins, products ofdnaA locus.

Severalevents occur at the site oforiginofreplication -

a. Appropriate initiation proteins must recognize the specific origin site.

b. The site must be opened by attachment ofhelicase (dnab). These

unwind the DNA at the Y-junction. The opened double helix is stabilized

by the single strand binding proteins (ssb)

c. The replication fork must be initiated in both directions, involving

continuous and discontinuous DNA replication.

The enzyme Primase, creates the RNA primers and together with the

helicase forms the primosome, which attaches to the lagging strand

template.

As the primosome moves along the replicating fork, they form the RNA

primers which are used by DNA polymerase III to synthesize the Okazaki

fragments.


41/50


42/50


43/50


44/50

DNA polymerase III holoenzyme is a large protein composed of 10 subunits

viz; , , , , , , , , , .

Polymerase cycling:

The subunits of DNA polymerase III allow the polymerase to move off and

on the DNA of the lagging strand template as Okazaki fragments are

completed.

Supercoiling:

Topoisomerase are enzymes that can bring about the supercoiling of the

DNA.

Supercoils could be positive ( when the circular duplex winds about itself in

the same direction as the helix twists (right handed))or negative (duplex

winds about itself in the opposite direction as the helix twists (left handed)).

Type I Topoisomarases break one strand of the double helix and while

binding the broken ends, pass the other (intact) strand through the break.

The break is then sealed.

Type II Topoisomerases (DNA gyrase in E.coli) break both the strands of the

double helix and pass another double helix through the temporary gap.


45/50


46/50


47/50


48/50

Replication Structures

Rolling Circle model:


49/50

D loop model:


50/50

Eukaryotic DNA replication:

These have multiple origins of replication,

resulting in formation of bubbles.

In yeast, DNA replication initiates at sites called

autonomously replicating sequences (ARS).

Six proteins form a complex called the origin

recognition complex (ORC).

ch 9 chemistry of gene

Documents