DNA REPLICATION: Definition
During cell division, DNA replication occurs. It’s bidirectional, discontinuous, and semi-conservative (semi-conservative mechanism was demonstrated by Meselson and Stahl in 1958).DNA replication is a critical mechanism for an organism’s cell development, repair, and reproduction.
OVERVIEW OF DNA STRUCTURE
James Watson and Francis Crick established the structural model of DNA for the first time. They discovered that DNA has two linked DNA strands with complementary nucleotide sequences, forming a Double-Helical structure.
1. The Double-stranded DNA molecule is made up of two spiral nucleic acid chains that are twisted together to form a Double Helix. The twisting is responsible for the DNA’s compactness.
2. DNA is made up of millions of nucleotides, which are deoxyribose sugar molecules with a phosphate group and a nucleobase connected to them.
3. Each Nucleotide is base paired with a complementary nucleotide on the opposite strand, e.g., Adenine (A) paired with Thymine (T) or Guanine (G) paired with Cytosine (C) – via Hydrogen Bonds (A=T and G= C).
4. Phosphodiester linkages bind nucleotides together in strands, generating a sugar-phosphate backbone. The bond is formed by a linkage between the 3′ (three prime) carbon atom of the deoxyribose sugar and the 5′ (five prime) carbon atom of another sugar on the following nucleotide,
5. DNA is tightly packed into tight coils known as chromatins to fit into the nucleus.
6. During cell division, the procedure occurs (Interphase – S phase).
7. During cell division, chromatins compress to produce chromosomes. The chromatins loosen up before DNA replication, allowing the replication machinery access to the DNA strands.
8. DNA replication necessitates the cooperation of a large number of proteins/enzymes.
9. Nucleotide triphosphate polymerization, i.e. adding nucleotides to the 3’end (5′ to 3′), is catalyzed by DNA polymerase and DNA primase. Primer is added by Primase.
10. DNA helicases and single-strand DNA-binding (SSB) proteins, help open the DNA helix and prevent breakage and renaturation, respectively.
11. To seal together the discontinuously generated lagging strand DNA fragments, DNA ligase and an enzyme that destroys RNA primers are used.
12. DNA topoisomerases/gyrase, which can help with helical winding and DNA tension issues. The DNA clamp is a protein that keeps elongating DNA polymerases attached to the DNA parent strand (beta subunits of the holoenzyme – DNA polymerase).
DNA-Dependent DNA Polymerase:
With the help of other enzymes, it aids in polymerization and catalyzes the entire process of DNA replication. There are three types of DNA polymerase:
DNA Polymerase I:
This enzyme is involved in DNA repair. It participates in three different activities:
1. The activity of the 5′-3′ polymerase
2. The activity of the 5′-3′ exonuclease
3. Exonuclease activity 3′-5′ proof reading
It bridges the gap between the strand pieces that are lagging.
DNA Polymerase II is in charge of primer extension as well as proofreading.
DNA Polymerase III is in charge of DNA replication in living cells. It is made up of two subunits, one for each strand. The true polymerase is the alpha subunit.
Helicase: Helicase is an enzyme that breaks the hydrogen bonds between DNA strands to unzip them. As a result, it contributes to the creation of the replication fork.
Ligase: This enzyme forms phosphodiester linkages between 3’OH and 5′ PO4 to connect the fragmented DNA strands.
Primase: This enzyme aids in the production of complementary short RNA primers to the DNA template strand.
Single-stranded Binding Proteins: These proteins bind to single-stranded DNA and prevent secondary structures from developing, keeping the strand unwound. The DNA polymerase III holoenzyme dimer, the primosome, and the DNA helicases are thought to be physically linked in vivo in a huge complex known as a replisome. A paternal duplex’s strands A daughter duplex is formed when DNA serves as a template for the synthesis of a daughter strand and stays base-paired to the new strand. In the 5′ to 3′ direction, new strands develop. Replication starts at a point known as the genesis of replication. Multiple replication sources are found in each eukaryotic chromosomal DNA molecule.DNA replication is a bidirectional process in which a replication fork arises at an origin and strand synthesis proceeds in opposing directions, with both template strands duplicated at the replication fork. One daughter strand (the leading strand) is constantly extended at a replication fork. The other daughter strand (the lagging strand) is made up of Okazaki fragments generated every few hundred nucleotides, forming a series of discontinuous Okazaki fragments. The pyrophosphate produced by the hydrolysis of the dNTPs provides energy for polymerization.
Leading strand
The leading strand is a strand of nascent DNA that is generated in the same direction as the replication fork that is expanding. The replication of DNA in this manner is never-ending.
Lagging strand
The lagging strand is a strand of nascent DNA whose synthesis is in the opposite direction as the replication fork grows. Replication of the lagging strand is more difficult than that of the leading strand due to its orientation. The lagging strand is made up of short, distinct segments. A primase “reads” the template DNA on the lagging strand template and starts the production of a short complementary RNA primer. Okazaki Fragments are formed when a DNA polymerase stretches the primed segments. This type of DNA replication is sporadic. The RNA primers are then removed and replaced with DNA, and DNA ligase is used to fuse the segments of DNA. AT-rich sequences are found in all origins where the strands first divide.
Steps in the Replication of DNA
INITIATION
At the origin of replication, the two strands of DNA unravel. The helicase opens the DNA, and a replication fork forms at the spot where the strands separate. Single-strand binding proteins wrap the DNA surrounding the replication fork to prevent it from rewinding. Topoisomerase stops DNA from supercoiling and relieves tension. Primase, which is complementary to the DNA strand, synthesizes RNA primers. In prokaryotes, initiation occurs at a single location called OriC.In eukaryotic DNA, there are several origin sites. DNA is the major initiator protein in E.coli.This is the Origin Recognition Complex in yeast (ORC), Because A-T base pairs have two hydrogen bonds (rather than the three produced in a C-G pair) and are thus easier to strand-separate, initiator protein sequences tend to be “AT-rich” (rich in adenine and thymine bases). The formation of the origin recognition complex catalyzes the assembly of initiator proteins into the pre-replication complex in eukaryotes.
ELONGATION
At the end of the primers, DNA polymerase III begins adding nucleotides. With the production of Okazaki fragments in the lagging strand, the leading and lagging strands continue to elongate.
TERMINATION
The primers are removed, and the gaps are filled using DNA Polymerase I, then ligase is used to seal the gaps. Termination occurs when a termination site sequence in the DNA is reached, and a protein, the DNA replication terminus site-binding protein, Ter protein, an inhibitor of DnaB helicase that functions in tandem with a Tus factor, attaches to this sequence to physically cease DNA replication (forming a complex).
DNA Replication in Prokaryotes and Eukaryotes (Differences)
1. The replication of DNA in eukaryotes is similar to that of prokaryotes, with a few exceptions.
2. All prokaryotic chromosomes are circular, as are many bacteriophages and viral DNA molecules.
DNA Replication Regulation
1. In Eukaryotic cells, DNA replication regulatory systems are critical for cell cycle control.
2. During the S-phase of each cell cycle, the eukaryotic genome is only replicated once.
3. The signal transduction pathway is the molecular method through which a cell’s growth factor signal is conveyed from the outside to the nucleus, causing the cell to start replicating and growing.
4. Protooncogenes are genes that encode components of the signal transduction pathway and can cause cancer if they are changed.
5. Eukaryotic DNA replication is regulated at different stages to guarantee that all chromosomes only replicate once every cell cycle.
6. Protein phosphorylation regulates the cell cycle, ensuring that pre-RC assembly can only take place in the G1 phase, while helicase activation and loading can only take place in the S phase.
7. DNA has ATP/ADP-binding and DNA-binding domains, while the origin has several DnaA binding sites. An active initiation complex can be produced at the origin when enough ATP-DnaA has accumulated in the cell, resulting in strand opening and recruitment of the replicative helicase.
8. DNA methylation and particular oriC-binding proteins directly govern oriC activity in Escherichia coli. Bacteria also have oriC-binding proteins that regulate them.
9. Proteins that induce ATP-DnaA hydrolysis, resulting in inactive ADP-DnaA, regulate DnaA activity. Initiation is further aided by the regulation of DnaA gene expression, which is conserved in bacteria.
10. CDK complexes in the S phase promote the start of coordinated DNA synthesis. Each chromosome is reproduced only once, thanks to the machinery. (CDK complexes are protein kinases that consist of a regulatory and catalytic subunit.) Cyclins are the regulatory subunits, whereas cyclin-dependent kinases (CDKs) are the catalytic subunits.
11.Cdc7-ASK (Activator of S phase Kinase), a new mammalian kinase, acts as a molecular switch for DNA replication upon entry into the S phase. This kinase is active specifically during the S phase and stimulates DNA replication by phosphorylating a key component of the replication complex, the DNA helicase.
12. The progression of cells from the G1 to S phase is regulated by several proto-oncogenes and tumor suppressor genes.