DNA Replication

DNA Replication

Each time a cell divides into two daughter cells, all the DNA moleculem be duplicated.
The process by which a DNA molecule produces its identical copies is described as
DNA replication.

It is a type of cell duplication or self reproduction of DNA, where two daughter
molecules are formed from a single DNA molecule.

Theoretically, three possible modes of DNA replication are possible. They are:

1. Dispersive replication:

The two strands of parent DNA break
randomly and produce several Pieces. These
pieces replicate and reunite to form new
daughter DNA molecules. These new DNA molecules
contain a mixture of old and new nucleotides
scattered along, the chains. The daughter molecules
can be described as hybrids. This mechanism is now

accepted nor proved experimentally.

2. Conservative replication:

After replication, one daughter DNA contains the original
two strands of the parent molecule. While the other
daughter molecule contains two newly synthesized
strands.this method is also not accepted.

3. Semi-conservative replication:

This method of DNA replication was proposed by Watson
and Crick. Because of specificity of base pairing, the
sequence of bases along one chain automatically
determines the basesequence along the other.

Half of the DNA is conserved i.e., only one strand is
synthesized and the other half of the original DNA
is retained.

The evidence for semi conservative replication of DNA molecules were provided by
Meselson and Stahl using ¹⁵N - a heavy isotope of ¹⁴N. 

J Cairns demonstrated the semi – conservative mode of replication of bacterial
chromosome using autoradiography technique.

Enzymes required for Replication:

Topoisomerase:

These enzymes can change the topological form or shape of DNA. Responsible
for initiation of the unwinding of the DNA. The tension holding the helix in coiled
and supercoiled structure can be broken by nicking a single strand of DNA.
DNA topoisomerases introduce a nick on only one of the DNA strands. This allows
the molecule to roatate around the phosphodiester bond on the opposite strand
as if it were a swivel. The topoisomerases introduce negative supercoils and relieve
strains in the double helix at either end of the bubble.

The Helicase:

Unwinds and unzips the DNA helix by breaking the hydrogen bonds between the
base pairs, thus allowing the two strands to separate.
The two strands very much want to bind together because of their hydrogen
bonding affinity for each other, so the helicase activity requires energy
(in the form of ATP) to break the strands apart.

DNA Polymerase:

Chief enzyme of DNA replication. Discovered by Kornberg in 1956. All the DNA
polymerase require the following:

1. A template DNA strand
   
   
2. A short primer (either RNA or DNA)
   
   
3. A free 3’ – OH in the primer.
   
   

They add one nucleotide at a time to the free 3’OH of the primer, and extend the primer
chain in 5’ → 3’ direction.

DNA polymerase is actually an aggregate of several different protein subunits, so it is
often called a holoenzyme. The holoenzyme also has proofreading activities, so that it
can make sure that it inserted the right base and nuclease (excision of nucleotides)
activities so that it can cut away any mistakes it might have made.

DNA polymerase I :

This enzyme was first purified by Kornberg in 1956. Hence, it is also called Kornberg
enzyme. This enzyme has three activities, which appear to be located in different
parts of the molecule.

A polymerase activity, which catalyses chain growth in the 5’ → 3’ direction

A 3’ → 5’ exonuclease activity, which removes mismatched base (DNA proof reading)

A 5’ → 3’ exonuclear activity, which degrades double stranded DNA (excision repair).

A exonuclease digests nucleic acids from one end (it does not cut DNA internally).

DNA polymerase II:

This enzyme repairs the damaged DNA. It has 5’ → 3’ polymerase and
3’→ 5’ exonuclease activities.

DNA polymerase III:

This enzyme is responsible for DNA replication in vivo. It has 5’ → 3’
polymerase and 3’ → 5’ exonuclease activities. DNA polymerase III is
a complex enzyme containing seven different polypeptides, and
5'to 3'of these polypeptides must be present for proper replicative

function. The 5' to 3' polymerase activity and the 5'to 3'exonuclease
activity are both present on the α polypeptide of DNA polymerase III.

The 3'to 5'proofreading activity of polymerase III is present on the
ϵ polypeptide.

Primase:

Is a part of an aggregate of proteins called the primeosome. The primase
(RNA polymerase) added to other proteins (forming a Primesome) makes
short pieces of RNA (RNA primers) that are recongnised by DNA polymerase III
to initiate replication.

As DNA polymerase III exhibits outstanding proofreading capabilities, which
prevent it to initiate a polynucleotide strand synthesis. Therefore it requires
a primer, a short piece of RNA (RNA primer) that it can recognize and elongate.
This RNA primer is eventually removed by RNase  and the gap is

filled in by DNA polymerase I.

Ligase:

Catalyze the formation of a phosphodiester bond given an unattached but
adjacent 3’OH and 5’ phosphate. This can fill in the unattached gap left
when the RNA primer is removed and filled in. The DNA polymerase can
organize the bond on the 5’end of the primer, but ligase is needed to

make the bond on the 3’ end.

The SSB  proteins (Single Strands Binding) stabilize the single strands thus
preventing them to zip back together and to form hairpin loops.
Single – stranded binding proteins are important to maintain the stability
of the replication fork. Single-stranded DNA is very labile, or unstable, so
these proteins bind to it while it remains single stranded and keep it from
being degraded.

Mechanism of DNA Replication

DNA synthesis is semiconservative (i.e., one of the parental DNA strands is conserved).

DNA replication begins at certain unique and fixed points called ‘Origin’ (ori)

Two enzymes DNA gyrase and DNA helicase, bind to the origin points and induce

the unwinding and separation of complementary strands of DNA double helix.

This separation is known as DNA melting.

Unwinding of DNA produces Y-shaped replication forks.

DNA polymerases proceed only in the 5’ to 3’ direction, DNA polymerase

require for their unction the presence of a :

i. Template DNA

ii. A primer (RNA or DNA, but only RNA is used in vivo).DNA polymerases can only

add nucleotides to the 3’OH group of a pre- existent primer

iii. Four deoxynucleoside triphosphates (dATP, dTTP, dGTP and dCTP), which are the

building blocks of DNA

As the two strands separate, the bases are exposed and RNA polymerase or

primase initiates transcription of the strand (3' to 5') and generates a 10 -60

nucleotide long RNA primer in 5' to 3' direction.

The free 3'- OH of this RNA primer provides the initiation point for the synthesis

of new DNA strand. DNA polymerase III adds deoxyribonucleotides to the 3'- OH group

of the last ribonucleotide of the RNA primer.

DNA polymerase I catalyzes the removal of the RNA primers by the concerted action

of its 5'to 3' exonuclease activity and its 5'to 3' polymerase activity.

DNA poly III progressively adds deoxyribonucleotides to the free 3'- OH of this growing

polynucleotide chain according to the base pairing rules (A=T; G=C). Consequently,

the replication of 3' to 5' strand of a DNA molecule proceeds continuously.

Because both strands of a DNA molecule are antiparallel, i.e., run in opposite directions,

this creates a problem. When the two strands unwind at the replication fork the leading

strands faces the DNA polymerase in correct 5’ to 3’ direction, so that the synthesis

of a long continuous complementary strand takes place.

On the lagging strand this is not possible; therefore, replication proceeds in a

discontinuous way, synthesizing short segments of DNA (always in the 5’→3’ direction)

called the okazaki fragments.

These segments are then joined together by the action of a DNA ligase. This short

discontinuous DNA segments were discovered by T. Okasaki, who exposed bacteria

to ³H -thymidine for a few seconds and found that fragments of DNA 1000 - 2000

nucleotides long. These fragments were called Okasaki fragemnts after their discoverer.

These are only 200 nucleotides long in eukaryotes.

DNA polymerases can only elongate a primer molecule; they cannot start a new chain

by themselves. This poses a problem when initiating each Okasaki fragment,

which is solved by synthesizing a short segment of RNA that acts as the primer.

Each Okasaki fragment starts with an RNA segment. RNA is used (instead of DNA)

because it immediately provides a ‘tag’ indicating that this part of the molecule has

not yet been subjected to the proofreading mechanism and must be replaced.

This RNA segment is subsequently removed by repair enzymes, the gap is filled

by DNA polymerase, and then joined to the neighbouring Okasaki fragment by a

DNA ligase to form a long polynucleotide chain.

The rate of movement of a replication fork in E.coli is about 10⁵  base pairs

per minute. In eukaryotes the polymerases are much less active and the rate

ranges from 500 to 5000 base pairs per minute.

In E.coli, the termination is signalled by specific sequences called ter elements. They

serve as binding site for protein Tus. The Tus protein binds to ter element and stops

helicase enzymes from unwinding DNA. This stops the movement of the replication fork.

Nucleic Acids

Nucleic acids are chain like macromolecules functioning in storage and transfer of genetic information. They are the major components of all cells, making up from 5 – 15% of their dry weight.

Although nucleic acids are so named because DNA was first isolated from cell nuclei, both DNA and RNA also occur in other parts of cells.

They are found in all living cells and viruses. They were first isolated in 1868, by a swiss scientist – Joseph Frederick Miescher from the nuclei of the pus cells on hospital bandages. He called them nuclein, because it came from nuclei and was something different from the protein. Altman (1889) coined the term nucleic acid, as it was found that nuclein was strongly acidic.

In 1930, A. Kossel demonstrated that nucleic acid on hydrolysis gave four nitrogen-containing compounds (adenine, guanine, cytosine and thymine; collectively called bases). P.A.T. Levene discovered for the first time that nucleic acids also contained a sugar molecule, which had a 5-carbon ring, and demonstrated that nucleic acids could be of two types, the deoxyribonucleic acid or DNA and ribonucleic acid or RNA

O.T. Avery, C.M. Macleod and M. McCarthy through transformation experiment indicated that DNA and not the protein is the genetic material. In 1952 A.D Hershey and M.J Chase using radioactive labeled phosphorous (³²PO₄) demonstrated that DNA is the sole genetic material.

Constituents of Nucleic Acids:

Chemical analysis have shown that nucleic acids are composed of the following 3 types of molecules:

1)      a pentose sugar

2)      a heterocyclic nitrogen base

3)      a phosphate group

Pentose Sugar:

Pentose sugar serve as building blocks of nucleic acids. The pentose sugar present is RNA is called D-ribose from which this nucleic acid gets its name. But DNA contains 2’-deoxy-D-ribose (simply deoxyribose). The oxygen atom present at the second carbon of ribose is missing in deoxyribose, giving its name 2’ – deoxyribose. The positions of carbon atoms of two pentose sugars are denoted as 1’,2’,3’,4’,5’ in order to differentiate them from the corresponding position in Nitrogen bases.

Nitrogen Bases:

The organic bases present in nucleic acids are heterocyclic compounds containing nitrogen in rings. Hence, they are also called as nitrogen bases. They are of two types: Purines and Pyramidines.

Pyrimidines:

            They have a six member ring containing two nitrogen atoms in place of carbon at position 1 & 3. The three pyrimidine bases : Thymine, Uracil, Cytosine contain a keto oxygen (= O) at position 2. In cytosine, an amino (-NH₂) group is present at position 4, in uracil another keto (=O) group is present at the fourth carbon, while thymine is essentially 5-methyl uracil i.e., a keto (=O) at position 4 and an CH₃ group at carbon 5.

All pyramidines, therefore, contain an –H atom at position 3, which is involved in their linkage with the 1C’ of pentose sugar.

Purines:

They have a six member pyrimidine ring, fused to a five member imidazole ring. Purines have 4 nitrogen atoms in place of carbon (C) at position 1,3ofpyrimidine ring and 7,9 of imidazole ring.

The nitrogen present at the 9^th position of purines participate in a covalent linkage with 1’C of the pentoses.

Adenine (A) and Guanine (G) are purines. In adenine, an amino (-NH₂) group is present at position 6. But in guanine, a keto (=O) group is found at position 6 and an additional –NH₂ group attached at the position 2.

Phosphate group:

Phosphoric acid (H₃Po₄) has 3 reactive hydroxyl groups (-OH) of which two are involved in forming the sugar – phosphate backbone of DNA. A phosphate moiety binds to the 5’C ov one and the 3’C of the other neighbouring pentose molecule of DNA to produce the phosphodiester (5’C – O – P –O – C 3’) linkage.

Nucleosides:

A nitrogen base combined with a sugar molecule is called as nucleoside. A base is linked with the pentose sugar molecule by a β – glycosidic bond.

The glycosidic linkage involves 1’C of the sugar and the nitrogen atom of N-9 (in purines) or N-3 (in pyrimidines) eliminating a molecule of water.

Nucleosides containing ribose sugar are called ribonucleosides, while those possessing deoxyribose sugar as deoxyribonucleosides.

Four common ribosides are adenosine, gauanosine, uridine and cytidine. Similarly, the four common deoxyribosides are deoxy-adenosine, deoxyguanosine, deoxycytidine and thymidine. (the name of pyrimidine nucleoside end with the suffix –dine, and those of purine end with suffix –sine)

Nucleotides:

The nucleotide is derived from a nucleoside by addition of one molecule of phosphate group. The phosphate molecule is linked with sugar molecule at carbon no. 5 or at carbon no.3. Correspondingly nucleotides will be called 5’p3’OH nucleotide and 3’p5’OH nucleotide. The four nucleotides found in DNA are deoxycytidylic acid, deoxythymidylic acid, deoxyadenylic acid and deoxyguanylic acid. Similarly, the four nucleotides found in RNA are cytidylic acid, uridylic acid, adenylic acid and guanylic acid.

Structure of DNA:

•      In 1953 using critical information from Rosalind Franklin and Linus Pauling, Watson and Crick determined the double helical structure of DNA  in 1953. Their double-helix model of DNA structure was based on two major kinds of evidence.

•      1. When the composition of DNA from many different organisms was analyzed by E. Chargaff and colleagues, it was observed that the concentration of thymine was always equal to the concentration of adenine (A =T) and the concentration of cytosine was always equal to the concentration of guanine (C = G).

This strongly suggested that thymine and adenine as well as cytosine and guanine were present in DNA with some fixed interrelationship. It also necessitated that the total concentration of pyrimidines (C + T) always equal to total concentration of purines ( A + G)

There is an equivalence between the bases carrying amino groups at the 6 or 4 positions (A + C) and those carrying keto groups at these positions ( G + T).This is popularly known as Chargaff’s rule.

Chargaff’s rule suggests that A is always paired with T. G always paired with C.

2. When X rays are focused through isolated macromolecules or crystals of purified molecules, the X rays are deflected by the atoms of the molecules in specific patterns, called diffraction patterns, which provide information about the organization of the components of the molecules. These X-ray diffraction patterns can be recorded on X-ray sensitive film.

Watson and Crick had available X-ray crystallographic data on DNA structure from the studies of M.H.F Wilkins, R. Franklin. 

On the basis of Chargaff’s chemical data, Wilkins and Franklin’s X – ray diffraction data, and inferences drawn from model building, Watson and Crick proposed that DNA exists as a double helix.

The main features of Watson – Crick model of DNA are:

1.      A DNA molecule is made up of two polynucleotide chains or strands which are coiled about one another in a spiral.

2.      Each polynucleotide chain consists of a sequence of nucleotides linked together by phosphodiester bonds between their sugar and phosphate residues.

3.      The two strands of a DNA molecule are oriented antiparallel to each other i.e., one strand runs in

5’      3’ direction, while the other strand runs in 3’      5’direction. This opposite polarity of the strands is very important in considering the mechanism of replication of DNA.

4.      The anitparallel orientation is essential as the two polynucleotide strands are held together in their helical configuration by hydrogen bonding between bases in opposing strands, the resulting base-pairs being stacked between the two chains perpendicular to the axis of the molecule like the steps of a spiral staircase.

5.      The base – pairing is specific, adenine present in one strand of DNA is always paired with thymine located opposite to it in the other strand. Similarly guanine located in one strands is always paired with the cytosine located opposite to it in the other strand.

6.      Thus, all base-pairs consists of one purine and one pyrimidine. This specificity of base-pairing results from the hydrogen – bonding capacities of the bases. Adenine and thymine form two hydrogen bonds, and guanine and cytosine form three hydrogen bonds.

7.      Once the sequence of bases in one strand of a DNA double helix is known, the sequence of bases in the other strand is also known because of the specific base-pairing. The two strands of a DNA double helix are thus said to be complementary (not identical). The formation of hydrogen bonds between A and T, and between G and C is thus known as Complementary base pairing.

8.      This property, complementarity of the two strands that makes DNA uniquely suited to store and transmit genetic information.

9.      The two strands of a DNA molecule are coiled together in a right-handed helix forming the DNA double helix. The diameter of this helix is 20 A^o.

10.  The pitch i.e., the length of helix required to complete one turn, is 34 A^o.  Thus, each turn contains 10 equally spaced base pairs. The distance between successive base pairs is 3.4 A^o and the angle between them is 36^o

11.  Each turn of DNA molecule include one major (wider) groove and one minor (narrow) groove along the phosphodiester backbone. Proteins interact with DNA at these grooves.

12.  The high degree of stability of DNA double helices results in part from the large number of hydrogen bonds between the base- pairs(even though each hydrogen bond by itself is quiet weak), and in part from the hydrophobic bonding between the stacked bas e-pairs. The planar sides of the base-pairs are relatively non polar and thus tend to be water insoulube or hydrophobic. This hydrophobic core of stacked base-pairs contributes considerable stability to DNA molecules present in the aqueous protoplasms of living cells.

Why DNA is helix

      The tendency towards a helix comes from the stacking of the individual bases on the top of one another. Both sugar and phosphate which constitute the backbone are quite soluble in water.   

       However, the DNA bases which are in the middle of the helix are relatively hydrophobic and insoluble.

    

      Since, the bases are flat, they stack on top of each other in order to form a more hydrophobic environment. The bases twist slightly in order to maximize their hydrophobic interactions with each other and it is this twisting of the stacked bases that gives rise to a helix. Thus, the reason for a helix in DNA is primarily due to hydrophobic stacking interactions of the bases.

Types of DNA

B-DNA:

The vast majority of the DNA molecules present in the aqueous protoplasms of living cells almost certainly exists in the Watson-Crick double-helix form. This is the B-form of DNA. The B-form is the conformation that DNA takes under physiological conditions i.e., in aqueous solutions containing low concentrations of salts.

B-DNA shows clockwise (right-handed) helix structure with 10 base-pairs per turn and has a pitch of 34A A^o.

However, DNA is not a static, invariant molecule. DNA molecules exhibit a considerable amount of conformational flexibility. The structures of DNA molecule change as a function of their environment.

A-DNA:

A-DNA for any sequence is favoured under dehydrating conditions ( 75% relative humidity). It appears that at least four purines (ex. GAGGGA) or pyrimidines in a row are enough to set up a local A-DNA helix. A- DNA helix is bit wider than B-DNA, and this is mainly due to the fact that the base pairs stack nearly on top of each other in B-DNA but stack a little off-centre in the A-conformation. There are about 11 base pairs per turn and with a pitch of 28 A^o.

Z-DNA:

It is an left handed helix. The backbone is not smooth helix, but is irregular and zig-zag in shape, hence its name. Alternating purine-pyrimidine can form, left handed Z-DNA. Z-helix is narrower than A and B –Conformation. It has 12 base-pairs per turn and with a pitch of 45 A^o.

Whether a DNA sequence will be in the A-, B- or Z-DNA conformation depends on 1. Ionic or hydration environment, A-DNA is favoured by low hydration, where as Z-DNA can be fovoured by high salt.

The second condition is the DNA sequence – A-DNA is favoured by certain stretches of purines or pyrimidines, where as Z-DNA can be most readily formed by alternating Purine-Pyrimidine steps.

The second condition is the presence of proteins that can bind to DNA in one helical conformation and force the DNA to adopt a different conformation.

In living cells, most of the DNA is in a mixture of A- or B-DNA conformation, with a few small regions capbable of forming Z-DNA.

Character	A-DNA	B-DNA	Z-DNA	C-DNA
Coiling	Right handed	Right handed	Left handed	Right handed
Pitch	28 Ao	34 Ao	45 Ao	31 Ao
Base pairs per turn	11	10	12	9.33
Diameter	23 Ao	20 Ao	18 Ao	19 Ao

Functions of DNA:

DNA performs two functions, they are:

I.                   Autocatalysis: the process of duplication of a single DNA molecule into two daughter DNA molecules is called autocatalysis or replication. DNA replicates by semi-conservative mechanism i.e., the daughter DNA molecules show one old or parent strand and one newly synthesized strand.

II.                Heterocatalysis: DNA promotes the synthesis of proteins and regulates bio-chemical reactions of cell. In this process the DNA templates transfer genetic message to mRNA by a process called transcription. mRNA helps in the synthesis of proteins.

DNA     Transcription         mRNA       Translation           Protein