DNA Replication

DNA Replication

DNA Replication

DNA Replication is the process by which DNA makes a copy of itself during cell division. It is semi conservative replication as both the copies contains one old strand and one new strand.

Basic requirement of replication

  1. Substrates : The four deoxynucleoside triphosphates ; dATP(deoxy-adenosine triphosphate) , dGTP(deoxy-guanosine triphosphate) , dCTP(deoxy-cytidine triphosphate) and dTTP(deoxy-thymidine triphosphate) are needed as substrate for DNA synthesis.
     
  2. Template : A template is required to direct the addition of the approptiate complementary deoxynucleotide to the newly synthesized DNA strand. In semi conservative replication , each strand of parental DNA serves as template. Then , each template strand and newly synthesized complementary strand serve as the DNA in daughter cells.
     
  3.  Primer : DNA synthesis cannot start withour primer , which prepares the template strand for the addition of nucleotides. Primase enzyme synthesized RNA primer.
     
  4. Enzymes :
    • DNA Helicase : It breaks the hydrogen bond formed between the bases of nucleotides , helps in unwinding of helix.
    • Topoisomerase : It is responsible for causing nick in the DNA strand to release the tension created during unwinding of DNA.
    • DNA Polymerase : Catalyses leading and lagging strand synthesis by adding complementary nucleotides in the template DNA.
    • DNA Ligase : It seals the gaps in the synthesized DNA (i.e bind the fragments of DNA strand together.
    • Repair Enzymes : It cuts off the wrong base of nucleotides and rejoin the correct base in newly formed DNA.
    • SSB Proteins : These protein molecules which attach tightly to the exposed single stranded DNA in order to stabilize the single stranded DNA long enough for replication.

Mechanism of Replication

  1. Initiation of DNA replication : DNA consist of many replication units called replicons . Replication starts at replicon known as origin or site of replication ( Ori-Site). In prokaryotic cell ,there is only one ori-site where as in eukaryotic cell it is more than one.
     
  2. Activation of deoxyribonucleotides : Inactive deoxyribo-nucleotide monophosphate present in nucleoplasm are changed into active deoxyribo-nucleotides triphosphate with the help of enzyme phosphorylase and ATP. This process is called phosphorylation.
    dNMPs
    dNTPs + H3PO4

     
  3. Unwinding of DNA helix : The unwinding of the DNA double helix takes places by the help of helicase enzymes which breaks the hydrogen bonds formed between bases of nucleotides by using energy ATP . Each unwinding parental DNA strand acts as a template DNA strand . When two strands unwind and separate incompletely , they form a Y- shaped structure known as replication fork . SSB protein stablized each separated strands .

    As DNA two strands are separated , DNA topoisomerase cut the phosphodiester link of DNA one strand of helix to prevent supercoiling thus allows the helix to rotate around the remaining unbroken strand . The enzyme then quickly reseals the link.

DNA Replication Fork

 

  1. Initation of DNA synthesis : DNA synthesis requires a primer . The primer are the short piece of RNA which is synthesized by RNA polymerase called primase.
     
  2. Chain Elongation : Once primer is formed , DNA replication beigns with the help of DNA polymerase III (in prokaryotes ) and DNA polymerase (in eukaryotes). Nucleotide chain formation proceeds by adding activated deoxyribonucleotides one at a time to the 3′ -OH end. A pair with T and G pair with C . New bases are only added in 5′ to 3′ direction .

    Replication is continuous in one template strand , single primer is responsible for the formation of whole strand . Hence, new strand is formed which is called leading strand. However, replication is not continuous in other template , more than one primer are responsible for the formation of whole strand . As a result , many smaller segment are formed called Okazaki fragment . The final gap between newly synthesized Okazaki fragment are join together by DNA ligase enzymes which form a complete daughter strand . Such strand is called lagging strand.

    After nucleotides chain formation is complete , RNA primer is removed by enzyme exonuclease and the gap is filled by complementary bases by DNA polymerase I .

     
  3. Termination : Termination sequences direct termination of replication . A specific protein , Termination Utilization Substance(TUS) protein binds to these sequences and prevents the helicase in further unwinding DNA. This facilitates the termination of replication .
     
  4. Proof Reading and DNA Repair : In some cases, there is mis insertion of wrong base. The correction is made during proof reading by repair enzyme.

Special Thanks To : Professor Raghav Raj Giri

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