The process in which two new DNAs of exactly the same nature are made from a double helix DNA is called DNA replication. Bacterial circular DNA molecules can be replicated at 100,000 base pairs per minute. Replication of long DNA molecules in real cells can add 500-5000 base pairs per minute. Replication does not start at either end of a real cell’s DNA.
DNA replication scheme
1. Conservative hypothesis: In the conservative process, DNA sequences are separated into two. Each sutra acts as a mold and creates new sutras. Then the old sutras are placed together and the new sutras are placed together. As a result, two strands of DNA are formed. That is, two strands of one DNA are old and two strands of other DNA are new.
2. Dispersive hypothesis: In the dispersive process, DNA strands are broken into two. Then different amounts of old and new fragments combine to form two DNA molecules. This process is not acceptable.
3. Semiconservative hypothesis: The process in which two new DNAs are formed from one maternal DNA and each of the two new DNAs has a maternal source and a new source is called a semiconservative process. In 1957, scientist Stent first used the term semiconservative. In 1958 Mathew Meselson & Franklin Stahl proved the semiconservative hypothesis in E. coli bacteria. In 1960, Herbert Taylor demonstrated semiconservative replication by experimenting with bean plant root cells. In 1960, scientist Sueka demonstrated semiconservative processes in human HeLa cells. In 1961, scientist Symon demonstrated the semiconservative process in the algae Chlamydomonas.
Meselson-Stahl’s test
Meselson and Stahl (1958) grew E. coli bacteria in a culture medium enriched in heavy isotopes of 14N and 15N. After several generations, the DNA of E. coli bacteria is labeled with 15N. The 15N enriched bacteria were then transferred back to the 14N medium. The next generation showed that the newly formed bacterial DNA double helix had one strand of 14N and the other strand of 15N. That is, one old formula and the other new formula of the double helix. This proves that DNA replication occurs in a semi-conservative manner.
During replication
Replication occurs in the interphase or S phase of the cell cycle and in the leptin subphase. Primitive cells contain circular DNA and have no ends or centers. Replication starts anywhere in the progenitor cell and replication forks move in both directions and meet at the midpoint. Replication of circular DNA is rapid and can add up to a million base pairs per minute. A cell’s circular DNA has two ends, and replication begins at multiple sites between the strands. Replication of circular DNA is slow and can add up to 500-5000 base pairs per minute. Replication of E. coli takes 20-30 minutes. Replication in animal cells takes 1.4-24 hours. Replication can start at 50,000 sites in Dosophila.
Steps of DNA replication
DNA replication is the most beneficial process in the living world. True cell replication is a complex process. The process can be discussed under three headings. Double helix separation, complementary strand formation and new DNA formation.
1. Separation of the double helix
(i) At the beginning of replication, base pairs are released at specific sites in the DNA and bubble-like ori points or replicon points or initiation points are formed. Ori spots are usually formed at sites where adenine and thymine are abundant in DNA. Because adenine and thymine have two hydrogen (A=T) bonds. Adikosha has one and Prakharkosha has multiple ori points.
(ii) First the helicase enzyme binds to the Ori point and starts unwinding the double helix. The helicase enzyme then uses energy from ATP to break the hydrogen bond. A Y-shaped structure is formed at the point where the two strands diverge. This is called replication fork.
(iii) After opening the patch, the two strands separated due to tension or attraction tend to gather again by re-adjusting the patch. The enzyme topoisomerase cuts the strand near the replication fork. As a result, the attraction of forming patches and gathering of the formula is lost. In primary cells, gyrase enzyme breaks down the attractive pull of formation and aggregation of sutra patches. The severed formula is then reattached.
(iv) The two formulas are complementary to each other. So it wants to re-attach by forming a hydrogen bond. Single Strand Binding Protein (SSBP) does not allow hydrogen bonds to form. As a result the two formulas cannot be rejoined.
(v) Replication forks move in opposite directions and balloon or eye-like structures are formed in the intervening space. It is called Replication eye or Replication bubble. Many replication bubbles are formed simultaneously. The bubbles elongate and coalesce, separating the two sutras. The separation of the two strands of DNA is called denaturation.
2. Creating complementary chains
(i) RNA primase enzyme uses the formula two as a template and creates smaller primers. The R3 end of the primer has a free -OH group.
(ii) DNA polymerase enzyme adds new nucleotides to the 3 -OH end of the primer. Nucleotides tend to join 5-3 carbons. As a result, two new sources started to emerge. One is the leading formula and the other is the lagging formula.
(iii) The strand that grows towards the replication fork is called the leading strand. Leading formulas continuously create counterpoints. The strain that grows in the opposite direction of the replication fork is called the lagging strain. The lagging formula produces unpaired segmental counterparts.
(iv) Each term of the lagging formula is called Okazaki. Okazaki of protocells is composed of 1000-2000 nucleotides and Okazaki of true cells is 100-200 nucleotides. Japanese scientist Reiji Okazaki and his wife Tsuneko Okazaki discovered it.
3. Creation of new DNA
(i) Exonuclease enzyme removes the primers complementary to the new sequence and the empty sites are filled with complementary nucleotides.
(ii) Incorrect nucleotides are removed by nuclease enzymes and correct nucleotides are added by DNA polymerase enzymes. This mismatch repair (MMR) is called DNA proof reading. Only one in 1,000 genes in humans can be a mismatch.
(iii) Ligase enzyme joins Okazaki. Purine and pyrimidine bases are joined by hydrogen bonds. As a result, two new DNAs are created.
replication complex
During DNA replication, some enzymes and proteins join near the replication fork to form complex molecular structures. It is called replisome or replication complex. The main components of replisome are-
1. DNA Helicase: The DNA helicase enzyme unwinds the DNA double helix at the replication fork.
2. DNA Polymerase: DNA polymerase enzyme joins nucleotides to form a complementary strand directed 5→3. DNA proof reading.
3. Topo-Isomerase: Topo-isomerase enzyme frees DNA from super coiled state.
4. DNA Clumps: DNA clumps inhibit maternal elongation of DNA.
5. DNA gyrase: The enzyme DNA gyrase creates supercoils (coils) at the end of DNA replication.
6. DNA ligase: The enzyme DNA ligase joins the Okazaki fragment to the complementary strand. Stops chain DNA sequence elongation.
7. SSBP: Single Strand Binding Proteins (SSBP) prevent single strand DNA molecules from refolding back into the double helix state.
8. Primase: Primase enzyme joins the RNA primer to the ends of the chain.
9. Telomerase: The telomerase enzyme adds nucleotides to telomeric DNA-molecules in eukaryotic chromosomes.
10. Exonuclease: Exonuclease enzyme removes the primers from the chain.
11. Nuclease: Nuclease enzymes remove incorrect nucleotides from complementary primers or strands.
Biological significance of DNA replication
1. Transcription: The role of replication for transcription is undeniable.
2. Transfer of Hereditary Traits: Replication helps in transferring the traits of an organism from one generation to another.
3. Rearrangement of Genes: New arrangements of genes occur in DNA molecules during replication.
4. Genetic Variation: Replication helps in creating genetic variation in organisms.
5. Manifestation of characteristics: Manifests all the characteristics of the organism.
6. Biological signals: Replication acts as a transmitter of biological signals in the organism.
7. Exact Replication: Exact copies are created through replication.
8. Protein synthesis: Replication plays a role in the synthesis of specific proteins for the cell.
9. Mutation: Changes in the structure of an organism through mutation.
10. Genetic map making: It has role in making chromosome map.
11. Genetic Studies: Introduction to genetic studies.
12. Formation of RNA: All types of RNA are formed from DNA.
13. Pathogenesis: Mismatch repair (MMR) causes colon cancer and skin diseases in humans.
