Fundamentals of Molecular Biology - V.I. Rezyapkin 2009

Replication
Proteins and Enzymes Involved in DNA Replication

Numerous Proteins and Enzymes are involved in METABOLISM/36.html">DNA Replication. Let us examine their main representatives. DNA polymerases

DNA polymerases drive DNA Synthesis. The substrates for these enzymes are dNTPs: dATP, dGTP, dCTP, and dTTP. The general equation for the reaction catalyzed by DNA polymerases is as follows:

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DNA polymerases sequentially add NUCLEOTIDES to the 3’-end of a polynucleotide chain:

The Synthesis of the new chain proceeds in the 5’-to-3’ direction.

A DNA polymerase can only elongate an existing DNA chain; it is incapable of initiating DNA synthesis de novo and requires a primer to begin its work. A DNA or RNA fragment can serve as a primer (Fig. 2.1). Furthermore, a DNA polymerase can only extend a chain in the presence of a template strand (Fig. 2.1). Nucleotides are added to the primer in accordance with the base-pairing rules: thymine is always incorporated opposite adenine, and cytosine opposite guanine (Fig. 2.1).

Fig. 2.1. DNA polymerase extends the chain in the 5’→3’ direction.

For this process, it requires both a template and a primer.

DNA polymerases are capable of copying any DNA strand, meaning they are not specific to The nucleotide sequence of the template. The rate of the DNA polymerase reaction ranges from 50 nucleotides per second in eukaryotes to 500 nucleotides per second in prokaryotes.

The primary task of DNA polymerases is to produce an accurate copy of the template. Consequently, they check the complementarity of each nucleotide twice: before incorporating it into the growing chain and before incorporating the next nucleotide. A new phosphodiester bond is formed if the incoming nucleotide is complementary to the template. If a non-complementary nucleotide is incorporated, it is removed via the 3’→5’ exonuclease activity of the DNA polymerase, and only after this erroneous nucleotide is excised does the DNA polymerase continue to elongate the DNA chain (Fig. 2.2).

Fig. 2.2. DNA polymerase removes a non-complementary nucleotide and then resumes DNA synthesis

Primase

Primase catalyzes the template-directed synthesis of a short RNA primer in the 5’→3’ direction. This primer is then utilized by DNA polymerase to initiate DNA synthesis (Fig. 2.3).

Fig. 2.3. Primase synthesizes An RNA primer, which is used by DNA polymerase for DNA synthesis

DNA ligase joins the 5’-phosphate and 3’-hydroxyl groups of adjacent nucleotides, resulting in The formation of a phosphodiester bond that seals the nick (Fig. 2.4).

Fig. 2.4. DNA ligase seals the nick

DNA helicase unwinds the DNA double helix, utilizing the energy of ATP Hydrolysis. Its action creates a Replication fork (Y) consisting of a double-stranded DNA region and two single-stranded branches (Fig. 2.5).

Single-stranded DNA-binding proteins (SSB proteins) exhibit a high affinity for single-stranded DNA and prevent the re-formation of The Double Helix (Fig. 2.5). Topoisomerases relieve the torsional strain generated by the unwinding of the DNA double helix by transiently cleaving and subsequently rejoining the DNA strand (Fig. 2.5).

Fig. 2.5. Roles of DNA helicase, SSB proteins, and topoisomerase