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Introduction. Type of DNAs. Modes of DNA Replication. Enzymes used in DNA Replication. Stages of DNA Replication. Comparison of Eukaryotic & Prokaryotic Replication.

References.

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DNA is a double helical structure. There are two polynucleotide chain coiled about one another in a spiral. Each polynucleotide chain consists of a sequence of nucleotides linked

together by phosphodiester bonds. The two polynucleotide strands are held together in their helical

configuration by hydrogen bonding between bases in opposing strands. The base-pairing is specific; adenine is always paired with thymine,

and guanine is always paired with cytosine. The two strand of a DNA double helix are complementary (not identical). Complementarity of two strands, that makes DNA uniquely suited to store

and transmit genetic information. The base pair in DNA are stacked 3.4 Ǻ apart with 10 base pair per turn. The sugar-phosphate backbones of two complementary strands are

antiparallel .

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Replication of DNA is central to all biology. DNA replication is a process to produce new

DNA molecules that have the same base sequence.

Occurs during interphase of the cell cycle DNA replication is semi-conservative.

◦ The parent DNA strand separates into two◦ Each strand serves as a template for new

complementary strands.◦ The new double helix is half original.

DNA REPLICATION

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DNA molecule exhibit a considerable amount of conformational flexibility.

(It is different in different physiological conditions.) DNA has 3 major forms:- 1. B – form 2. A - form 3. Z – form

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COMPARISON B-form A-form Z-form Helix sense- Right handed Right handed Left handed

Base pair _ 10 11 12per turn

Vertical rise _ 3.4 2.56 3.7 Angstromsper base pair

Rotation per_ +36 +33 ‑30 degrees base pair

Helical diameter- 19 23 18 Angstroms

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MODES OF DNA REPLICATION

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Bacteria were grown in a medium containing nitrogen 15 (N15) for several generation.

If the medium contains no other nitrogen source, the E. coli will use N15 and incorporate it into their DNA.

Eventually, they will only have N15 . Once the E. coli had only N15 they were put into a growing

medium contain only N14.

N15 is heavier than N14 making new incorporation of nitrogen easy to distinguish.

The differences were measured according to the densities of the new strands.

The Meselson-Stahl Experiment

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Molecule of different density separated by equilibrium density gradient centrifugation.

Meselson and Stahl were able to distinguish between the three possible mode of DNA replication by following the changes in the density of DNA of cell grown on N15 and then transferred to N14 medium for various period of time. So it is also called density transfer experiment.

Meselson and Stahl took E. Coli and washed it to remove N15 medium. Then transfer them to N14 medium . After one generation of growth in medium containing N14 , had a density halfway between the densities of heavy DNA and light DNA. This is called hybrid.

After two generation half is hybrid and half is light. Conservative mode do not produce any hybrid. In dispersive they should observe a shift of DNA from heavy to

light. Which was not observed.

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Position of Enzymes and Proteins on DNA

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Helicase uses energy from the ATP to break the hydrogen bond holding the base pair together.This allow the two parental strands of DNA to begin unwinding and forms two replication fork.Two different helicase are involved for unwinding of each strand.It is coded by gene dna B.

Primase is an enzyme that copies a DNA template strand by making a RNA strand complementary to it.Primase synthesizes a short (about 10 nucleotides) RNA primer in the 5’ to 3’ direction.It is product of dna G gene.Primase activity requires the formation of a complex of primase and at least six proteins ( protein i , n , n’ ,n” plus the product of gene dna B and dna C ),the complex is called the primosome.

Helicase:-

RNA Primase:-

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Single-stranded DNA binding protein (SSB) binds to the single-stranded portion of each DNA strand, preventing the strands from reassociating and protecting them from degradation by nucleases.The SSBP binds DNA as tetramer and their binding exhibit cooperativity (i.e. the binding of one tetramer to adjacent segment of single stranded DNA).SSBP binded DNA replicate over hundred times faster than uncomplexed single stranded DNA.It helps to stabilize the extended single stranded template needed for polymerization. It prevent folding back of DNA.

Single Stranded Binding Protein(SSBP) :-

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This enzyme can change the state of supercoiling in a DNA molecules.

It relieves the tension that builds up during replication. It catalyze the formation of negative supercoil in DNA. It removes positive supercoils.

DNA polymerase I is involved in removing RNA primers in the processing of DNA after replication.

Both DNA polymerase I and III have the ability to "proofread" their work by means of a 3' 5' exonuclease activity.

DNA Polymerase I :-

DNA Gyrase (Type II Topoisomerase) :-

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DNA polymerase III is a complex enzyme containing 10 different polypeptides (α,ε,ɣ etc.) . All those polypeptides must be present for proper replicative function.

The 5’ to 3’ polymerase activity and the 5’to 3’ exonuclease activity both present on the α-polypeptides of DNA polymerase III.

The 3’to 5’ proofreading activity of polymerase III present on the ε -polypeptide.

The function of other subunit still uncertain. It catalyse the chemical reactions for polymerization of

nucleotides. DNA polymerase III begins synthesizing DNA in the

5’3’direction, beginning at the 3’ end of each RNA primer.

DNA Polymerase III :-

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DNA polymerase resembles a hand that grip the primer-template junction.

The speed of DNA synthesis is largely due to the processive nature of DNA polymerase.

IN case of DNA polymerase , the degree of processivity is defined as the average no. nucleotide added each time the enzyme binds a primer-template junction. It can be from few nucleotides to more than 50000 bases added per binding event.

Structure of DNA Polymerase III :-

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DNA ligase catalyses covalent closure of the resulting single-stranded “nick”.

DNA ligase joins Okazaki fragments, converting them to a continuous strand of DNA.

It is a protein that binds to terminator sequences and acts as a counter-helicase when it comes in contact with an advancing helicase.

The bound Tus protein effectively halts DNA polymerase movement.

Tus helps to end the DNA replication in prokaryotes.

Terminus Utilization Substance (TUS) Protein:-

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Initiation :-Occurs at the origin of replication.

Elongation:-Involves the addition of new

nucleotides based on complementarity of the template strand.

Termination:-Occurs at a specific termination site.

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Initiation- Occurs at the origin of replication.

ORI is the region where process of replication starts. This region is rich in A & T base pairs. It is easy to melt during the initiation.

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At ori there is presence of specific base sequences regions . One is 9 base pair long regions which have four repeats and

another is 13 base pair long have 3 repeats also called 13 mer region.

13 mers are A-T base pair rich regions.

Dna –A proteins binds to 9 b.p. region ,which cause change in 13 mer region and it start open up.

In 13 mer region due to presence of A-T b.p. ,it is easy to open as A-T b.p. has two hydrogen bonds as compared to C-G b.p. as it contain three hydrogen bond.

After this there is attachment of helicase and SSB proteins and other enzymes to carry on DNA replication.

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Elongation :- Involves the addition of new nucleotides based on complementarity of the template strand.

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Occurs in a 5’→3’ direction.◦ The 5’ end of the ‘new’ nucleotide attached to the 3’ end of the

nucleotide before it. DNA is unwounded and unzipped by the enzyme Helicase. Before DNA replication begins there must be RNA primer. The RNA primer is made by adding complimentary RNA nucleotides to the

lagging DNA strand by hydrogen bonding of the bases. RNA Primase then bonds the RNA nucleotides together. DNA polymerase III creates links between the nucleotides. It creates a strand that is complementary to the original strand. In eukaryotes replication takes place at several places on one double helix

at the same time. Once the double helix is unwound and unzipped the two parent strands

become the leading and lagging strands.

PROCESS OF DNA REPLICATION

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Replication is continuous. There are no fragments. DNA polymerase III adds nucleotides in the direction of 5’→3’.

◦ DNA polymerase only works in the direction of 5’→3’.

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The lagging strand runs from 3’ to 5’ of template strand. 1. After RNA primer is in place DNA nucleotides are added by DNA polymerase III. 2. Eventually, the segment of DNA will run into another RNA

primer. 3. The DNA segments are called Okazaki fragments. 4. Once Okazaki fragments are formed DNA polymerase I replaces the RNA primer with DNA nucleotides. 5. DNA Ligase links the fragments together. 6. In the lagging strand DNA replication is discontinuous. 7. DNA is replicated in segments that become joined together.

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In prokaryotes DNA replication terminates when replication fork reach specific ‘termination site’.The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex.A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region.The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertainThe two replication fork meet each other on the opposite end of the parental circular DNA.

Termination:-

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It is done by exonuclease activity of DNA Pol. III . The role of exonuclease become clear when it was determined that they have a strong preference to degrade DNA containing mismatch base pair.

The removal of mismatched nucleotide is facilitated by the reduced ability of DNA polymerase to add nucleotide an incorrectly base paired primer.

As for processive DNA synthesis , proofreading occurs without releasing the DNA from the polymerase.

When a mismatched base pair is present in the polymerase active site , the primer : template junction is destabilized , creating base pairs of unpaired DNA.

The newly unpaired 3’end moves from the polymerase active site to exonuclease active site.

The incorrect nucleotide is removed by the exonuclease. The removal of mismatched base allow the primer :template

junction to reform and rebind to the polymerase active site , enabling DNA synthesis to continue.

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Prokaryotes Eukaryotes1. It occurs inside the cytoplasm. 1. It occurs inside the nucleus.2. There is single origin of replication. 2. Origin of replications are numerous.3. DNA polymerase III carries out both initiation and elongation.

3. Initiation is carried out by DNA polymerase α while elongation by DNA polymerase δ and ε.

4. DNA repair and gap filling are done by DNA polymerase I.

4. The same are performed by DNA polymerase β.

5. RNA primer is removed by DNA polymerase I.

5. RNA primer is removed by DNA polymerase β.

6. Okazaki fragments are large, 1000- 2000 nucleotides long.

6. Okazaki fragments are short, 100- 200 nucleotides long.

7. Replication is very rapid, some 2000 base pairs per second.

7. Replication is slow, some 100 nucleotides per second.

8. DNA gyrase is needed. 8. DNA gyrase is not needed.

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