Professor A. K. GhoshDepartment of BiotechnologyEmail: aghosh@hijli.iitkgp.ernet.inStructure and Synthesis

Eukaryotic cells with a nucleus and various membrane bound organellesProkaryotic cells without a nucleus and membrane bound organelles Nucleus contains many chromosomes carrying genetic information in the form of nucleic acidNucleoid contains single chromosome carrying genetic information in the form of nucleic acid

Chromosome duplication (including DNA synthesis)CentromereSister chromatidsSeparation of sister chromatidsSister chromatidsOrganisation of chromosomes in the nucleus and status during division

Bacterial chromosomes are double-stranded DNA and are compacted into a nucleoid.

The length of the circular DNA molecule present in the chromosome of the bacterium Escherichia coli is about 1500 m.

Because an E. coli cell has a diameter of only 1 to 2 m, the large DNA molecule must exist in a highly condensed) and folded configuration

During bacterial replication chromosome replicates

Structure and composition of Nucleic acidsDeoxyribonucleic acid (a polymer of deoxyribonucleotides)

Ribonucleic acid (a polymer of ribonucleotides)

A nucleotide is made up of Pentose Sugar, Nitrogenous base and phosphate

BASES IN NUCELEIC ACIDS

TWO STRANDS OF DNA RUN ANTI - PARALLEL

Discovering the structure of DNA Structure was discovered in 1953 by James Watson and Francis Crick

Characteristics of three different DNA Form

A formB formZ form

Helical senseRight handedRight handedLeft handed

Diameter 26 A ( 2.6 nm)20 A (2 nm)18 A 9 1.8 (nm)

Base pair per helical turn1110.512

Helix rose per base pair2.6 A (0.26 nm)3.4 A (0.34 nm)3.7 A (0.37 nm)

Study Helix Stability with DNA Melting Curves

When DNA melts, the 2 strands come apart, and its absorbance in the UV region increases. Tm= temp. at which 50% of DNA is melted.

RNA StructureMore commonly, RNA is single stranded and can form complex and unusual shapes.

DNA vs. RNADNA Double HelixDeoxyribose sugarAdenine pairs with Thymine (A-T)Stays in nucleus RNASingle strandRibose sugarUracil replaces ThymineLeaves nucleus to do the work

DNA ReplicationProcess of duplication of the entire genome prior to cell division

Biological significanceextreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations

Replication rate in eukaryotes is slower resulting in a higher fidelity/accuracy .

Models of DNA replication Conservative- would leave the original strand intact and copy it.

Dispersive-would produce two DNA molecule with sections of both old and new along each strand.

Semiconservative would produce DNA molecule with both one old strand and one new strand.

The Meselson-Stahl experiment, which showed that DNA replicates semiconservativelyThis pattern could only have been observed if each DNA molecule contains a template strand from parental DNA. Thus it proves DNA replication is semiconservative

1955: Arthur Kornberg

Worked with E. coli. Discovered the mechanisms of DNA synthesis.

Four components are required:

dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5-triphosphates)(sugar-base + 3 phosphates)

2.DNA template and primer

DNA polymerase (Kornberg enzyme)4.Mg 2+ (optimizes DNA polymerase activity)

DNA PolymeraseAn enzyme that catalyzes the addition of a nucleotide to the growing DNA chain

Nucleotide enters as a nucleotide tri-PO4

3OH of sugar attacks first phosphate of tri-PO4 bond on the 5 C of the new nucleotidereleasing pyrophosphate (PPi)

55335533Watson/Crick proposed mechanism of DNA replication

Properties of DNA Polymerase Although this is the main Enzyme that helps DNA synthesis during DNA replication in E. coli and corrects mistaken base pairings, but it has some limitation in its action: It can not initiates DNA synthesis and requires a preformed or established polymer (small RNA or DNA) as primer to initiates DNA synthesis.

It can only adds nucleotides only in 5 to 3 direction.

It cannot unwind double stranded DNA

These limitations indicate other proteins and enzymes are required for DNA synthesis.

*Mechanism of DNA replicationDNA replication process can be devided in three steps:

initiation replication begins at an origin of replicationelongation new strands of DNA are synthesized by DNA polymerasetermination replication is terminated.

Initiation of replication starts at origin

DNA replication initiates at a particular region in DNA called origin of replication.

Prokaryotes single origin site E.g E.coli - oriC

Prokaryotes

Replication Origin Contains many A-T base pairs because easier to break 2 H-bonds than 3 H-bonds

Initiator Protein (Dna A protein) binds in the origin of replication and with the help of ATP unwinds DNA

1. Initiation takes place at OriC (which contains four 9 bp and three 13 bp AT rich repeat sequences) where the initiator protein ( DnaA) binds.

DnaA forms a complex of 30-40 molecules, around which the oriC DNA becomes wrapped. This process facilitates melting of three 13 bp AT-rich repeat sequence in presence of ATP and open double helix of DNA

In the open double helix then DnaB protein ( hellicase) carried by Dna C protein binds and continuously unwind the DNA.

4. To prevent rewinding of open single stranded DNA region, single stranded DNA binding protein (SSB) then binds.

Initiation

5. Removal of helical turns at the replication fork leads to the introduction of additional turns in the rest of the molecules in the form of positive supercoiling.

6. positive supercoiling is relaxed continuously by a type II topoisomerase called DNA gyrase.

7. DNA primase then attaches to the single stranded DNA and synthesizes a short RNA primer where DNA Polymerase III binds and initiates DNA synthesis ( 5 to 3 direction) in the leading strand of the replication fork.

Model for the formation of a replication bubble at a replication origin in E. coli and the initiation of the new DNA strand

From the replication bubble replication proceeds in both directions.

Lagging strand DNA synthesis

After the initiation of DNA synthesis in the origin of replication, DNA synthesis takes place continuously ( 5 to 3 direction) in that stand and is called leading strand

In the opposite strand of DNA, where DNA synthesis takes place little bit latter is called lagging strand.

In the lagging DNA strand, primase first bind and synthesize short RNA primer and then DNA polymerase III binds to synthesize DNA as short fragment ( 1000 2000bp). This short DNA fragment is called Okazaki fragment.

As the 3end of one okazaki fragment reaches 5 end ( RNA) of another Okazaki fragment, DNA polymerase III falls off , and DNA polymerase I binds and through its 5 to 3 exonuclease activity removes RNA primer and by its 5 to 3 polymerase activity synthesize DNA to fill the GAP.

The two DNA ends are then sealed or joined by DNA ligase

Thus lagging strand DNA synthesis takes place in a discontinuous manner

DNA Replication Fork

Model of DNA replication

Model of DNA replication

ElongationBoth leading and lagging strand primers are synthesized simultaneosly by DNA polymerase III : as a hetero dimeric complex, one half synthesizing the leading strand continuously and the other half synthesize lagging strand discontinuously.

Since two DNA strands run in opposite directions, tomaintain 5 to 3 directional polarity for DNA synthesis. Lagging strand of DNA make a loop through the enzyme molecules.

After synthesis of one Okazaki fragment it detaches from Polymerase III and a new single stranded lagging strand binds to DNA polymerase III and the process continues

Termination of Replication Terminus: is in approximately 180o opposite oirC containing several terminator sites (ter).

Tus protein: ter binding protein, an inhibitor of the DnaB helicase and can arrest the movement of the forks by binding to terminus.

Bidirectional replication of circular DNA molecules and segretgation Topoisomerase IV - A Type-II topoisomerase Cuts DNA and passes one duplex through the other.

Basic rules of replicationSemi-conservativeStarts at the originCan be uni or bidirectionalSemi-discontinuous Synthesis always in the 5-3 direction RNA primers required

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