MOLECULAR BASIS OF INHERITANCE - PART-IV|REPLICATION

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In this session Bio Strips Media takes through the important topics of Molecular Basis of Inheritance. It is one of the most important and scoring topics in Plus Two Biology Exam and NEET Exam. 

 

This lecture covers:

·         Replication of DNA

·         Enzymes involved in DNA replication

·         Mechanism of DNA replication

 

The content being discussed in this video will be helpful for those appearing for the Plus Two Biology Exam and NEET Biology Exam.

 

The information in this video is very useful to them and it helps those NEET aspirants to score maximum marks in Biology.

 

The Biology content in this video will be helpful for those candidates appearing for Kerala SET Zoology Exam, Kerala SET Botany Exam, KTET Natural Science Exam and various other Entrance Exams. 

 

DNA Replication

Replication is the process of formation of carbon copies or duplication of DNA.

Replication in eukaryotes occurs in the nucleus during the 'S' phase of the cell cycle. In prokaryotes replication takes place in the cytoplasm. Watson and Crick suggested that the two strands of DNA molecule uncoil and separate and each strand serves as a template for the synthesis of a new (complementary) strand.

Two daughter DNA molecules are formed from the parent DNA molecule and  these are identical to the parent molecule. Each daughter DNA molecule consists of one parent strand and one new strand. Since only one parent strand is conserved in each daughter molecule, this mode of replication is said to be e semi-conservative. Semiconservative replication of DNA a was found by Taylor (1957) in vicia faba using tritiated thymidine.

Meselson and stahl experimentally proved that DNA replicates by semiconservative method.

Meselson and Stahl experiment

E coli was grown in N15 medium having heavy isotope of Nitrogen for many generations. Labelled bacteria was transferred to fresh N14 medium. Replication in N14 medium. Further replication in N14 medium. DNA tested for heavy isotope of Nitrogen through density gradient centrifugation using caesium chloride (CsCl).

Results

DNA from bacteria that had been grown on medium containing N15 appeared as a single band.

After one round of replication the DNA appeared as a single band intermediate between that expected for DNA with N 15 and that expected for DNA with N14.

After second round of replication, DNA appeared as two bands one in the position of hybrid DNA (half N15 and half N14 )and the other in the position of DNA that contained only N 14.

Samples taken after additional rounds of replication appeared as two bands as in part c.

Conclusion 

DNA replication in E coli is semiconservative.

Replication of DNA

DNA replication in prokaryotes and eukaryotes starts from a specific point called origin of replication (ori) which is one in bacterial DNA and many in Eukaryotic DNA. The region where the helix unwinds and synthesis of new DNA starts is called the replication fork and the smallest unit of replication is called replicon.

Eukaryotic DNA are very large hence they represent several replicon. Bacterial DNA represents only one replicon.

The process involves a number enzymes and protein factors which are discussed below:

 DNA helicase:

 DNA helicase are atp-dependent unwinding enzymes which promote separation of the two parental strands by breaking hydrogen bonds between base pairs and establish replication Forks.

Single strand DNA binding proteins or SSBPs:

Behind the replication fork the single DNA strand is prevented from rewinding about one another by the action of SSB proteins.

Topoisomerases:

Topoisomerase cut and join one strand of DNA to facilitate  uncoiling. In Prokaryotes the function of topoisomerase is taken over by DNA gyrase.

DNA ligases:

Discovered by HG Khorana in 1967. Seal all the nicks in final replication product by forming phospho di ester bond between 5 prime phosphate and 3 Prime hydroxyl groups.

DNA polymerase or replicase:

These enzymes bring about the synthesis of new  polynucleotide chain.

Three different DNA polymerases are known in prokaryotes of which DNA polymerase 1 and 2 are meant for DNA repair and DNA polymerase 3 is meant for actual DNA replication.

DNA polymerase 1 enzyme is called as kornberg enzyme because it was isolated by Arthur kornberg around 1960.

Eukaryotes are found to contain 5 different types of polymerases namely alpha, beta,  gamma, delta and Epsilon.

Primer- forms short RNA primers required for initiation of replication.

The process is also an energy expensive process; deoxyribo-nucleotide tri-phosphates serve the dual purpose of (i) acting as a substrate and (ii) providing energy.

Mechanism of DNA replication

Helicase unwind the parental double helix. Molecules of single stranded binding protein stabilize the unwind template  strands. Topoisomerase releases tension of DNA strand. The initiation of DNA synthesis requires a RNA primer. The primer gross in 5 prime to 3 Prime direction. Initiation of replication occurs at 3 Prime end of the template. The enzyme DNA polymerase adds the nucleotides complementary to the DNA template in 5 prime to 3 Prime direction in the presence of ATP.

The enzyme synthesizes a new strand in continuous stretch on 3 Prime to 5 prime strand this strand is called leading strand.

The second new strand (lagging strand) is formed in short segments called okazaki fragments on the template strand with polarity 5 prime to 3 Prime. Okazaki fragments are later joined together by the enzyme DNA ligase.

The leading strand is synthesized continuously and the lagging strand is synthesized discontinuously so it is called semi discontinuous replication. RNA primer is removed by Exonuclease activity of DNA polymerase 1.