DNA Replicating enzyme/protein from E. coli and Human

 DNA Replicating enzyme/protein from E. coli and Human.

Enzyme/Protein	E. coli (Prokaryote)	Human (Eukaryote)
Helicase	DnaB	MCM 2- 7
ssDNA binding protein	SSB	RPA (Replication protein A)
Primase	Dna G	DNA Polymeraseα/ primase
Replicase	DNA Polymerase III	DNA Polymerase ε (on the leading strand)/ DNA Polymerase δ (on lagging strands)
Topoisomerase	Gyrase	Topoisomerase I, II
Processivity component	β-clamp	PCNA (Proliferating Cell nuclear antigen)
Clamp loader	γ- complex	RFC (Replication factor)

    

Question ( CSIR DECEMBER 2024) 

             Match the columns

Column X	Column Y
E. coli involved in replication	Functional eukaryotic orthologues
A.        DnaB	i.                 Polα/primase
B.      DnaC	ii.                Cdc 6
C.      Β clamp	iii.               PCNA
D.      Dna G	iv.               MCM complex

Which of the following options represents all correct matches between Column X and Column Y?

1.  A (i) ,     B (ii),       C (iii) ,    D ( iv) 
2. A(iv),      B  (i),      C(ii),        D(iii) 
3. A ( iv) ,   B  (ii),     C(iii),       D( i)
4. A( ii),      B (iv),      C (iii),     D (i)

Answer 3. A(iv), B(ii),  C(iii),  D(i)

Explanation of facts:( according to the Molecular biology of cell, ALBERTS, JOHNSON, LEWIS, RAFF, ROBERTS, WALTER,)

• Helicase: DNA helicases were initially identified as proteins that hydrolyze ATP while binding to single strands of DNA. This hydrolysis of ATP can cyclically alter the shape of a protein molecule, enabling it to perform mechanical work. DNA helicases use this principle to move rapidly along a single strand of DNA. When they reach a region of double helix, they continue their movement along their strand, effectively unwinding the helix at speeds of up to 1,000 nucleotide pairs per second. The two strands of DNA have opposite polarities. In theory, a helicase can unwind the DNA double helix by moving in a 5' to 3' direction along one strand or in a 3' to 5' direction along the other strand. In fact, both types of DNA helicases do exist. In the best-understood replication systems found in bacteria, a helicase that moves from the 5' end to the 3' end along the lagging-strand template plays a predominant role, for reasons that will become clear shortly.

• Single-strand DNA Binding (SSB) Proteins: Single-strand DNA-binding (SSB) proteins, also known as helix-destabilizing proteins, bind tightly and cooperatively to exposed single-stranded DNA without covering the nucleotide bases, which keeps them available for templating. These proteins cannot directly open a long DNA helix but assist helicases by stabilizing the unwound, single-stranded configuration. Additionally, their cooperative binding helps coat and straighten out the regions of single-stranded DNA on the lagging-strand template, preventing the formation of short hairpin helices that can easily occur in single-stranded DNA. These hairpin helices can interfere with the DNA synthesis process catalyzed by DNA polymerase.

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• Primase: DNA primase synthesizes short RNA primers on the lagging strand using ribonucleoside triphosphates. In eukaryotes, these primers are approximately 10 nucleotides long and are produced at intervals of 100-200 nucleotides on the lagging strand.
• Replicase: DNA Polymerases catalyze the synthesis of DNA.

• Topoisomerase: Topoisomerase is an enzyme that breaks the phosphodiester bonds in DNA strands. It acts as a reversible nuclease because it covalently attaches itself to a phosphate group in the DNA backbone, leading to the breakage of a phosphodiester bond. This process is reversible; once topoisomerase completes its function, the phosphodiester bond re-forms as the enzyme dissociates from the DNA. One type of topoisomerase, known as topoisomerase I, creates a transient single-strand break (or nick) in the DNA. This break in the phosphodiester backbone allows the two sections of the DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a pivot point. Any tension in the DNA helix drives this rotation in the direction that alleviates the tension. As a result, DNA replication can proceed with the rotation of only a short length of the helix—specifically, the segment just ahead of the replication fork.

• Processivity Component: A sliding ring holds a moving DNA Polymerase onto the DNA.  Example β-clamp in prokaryote andPCNA ( Proliferating Cell nuclear antigen) in eukaryote.

• Clamp loader: In DNA pol-III holoenzyme, two catalytic cores are linked by a clamp-loader (γ- complex in prokaryote ) that constitutes DNA Polymerase III.

 Summary :

 DNA replication occurs at a Y-shaped structure known as the replication fork. This process is facilitated by a self-correcting enzyme called DNA polymerase, which catalyzes the polymerization of nucleotides in a 5ʹ-to-3ʹ direction, accurately copying a DNA template strand. Because the two strands of a DNA double helix are antiparallel, continuous 5ʹ-to-3ʹ DNA synthesis can occur only on one strand at the replication fork, referred to as the leading strand. On the lagging strand, short segments of DNA must be synthesized through a “backstitching” mechanism. Since the self-correcting DNA polymerase cannot initiate a new chain, these lagging-strand fragments are primed by short RNA molecules, which are later removed and replaced with DNA.

DNA replication involves the coordinated action of several proteins, including:

1. DNA polymerase and DNA primase, which are responsible for nucleoside triphosphate polymerization;

2. DNA helicases and single-strand DNA-binding (SSB) proteins, which assist in unwinding the DNA helix for copying;

3. DNA ligase and an enzyme that removes RNA primers, which together seal the discontinuously synthesized lagging strand fragments;

4. DNA topoisomerases, which alleviate issues related to helical winding and tangling.

Many of these proteins work together at the replication fork to form a highly efficient "replication machine," coordinating the activities and spatial movements of individual components during the replication process.

REFERENCE:

1. Alberts B (1998) The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294.
2.  Kelch BA, Makino DL, O’Donnell M, et al. (2011) How a DNA polymerase clamp loader opens a sliding clamp. Science 334, 1675–1680.
3. Kornberg A (1960) Biological synthesis of DNA. Science 131, 1503 1508.
4. Li JJ & Kelly TJ (1984) SV40 DNA replication in vitro. Proc. Natl. Acad. Sci. USA 81, 6973–6977.
5.  Meselson M & Stahl FW (1958) The replication of DNA in E. coli. Proc. Natl. Acad. Sci. USA 44, 671–682.
6.  O’Donnell M, Langston L & Stillman B (2013) Principals and concepts of DNA replication in Bacteria, Archaea, and Eukarya. Cold Spring Harb. Lab. Perspect. Biol. 195, 1231–1240. 
7. Vos SM, Tretter EM, Schmidt BH, et al. (2011) All tangled up: how cells direct, manage and exploit topoisomerase function. Nat. Rev. Mol. Cell Biol. 12, 827–841.