Enzymes Involved in DNA Replication: Their Functions and Role in DNA Synthesis, Slides of Biotechnology

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Enzymes used in DNA Replication

This document holds the enzymes used in DNA replication, their pictorial representation and functioning.

DNA polymerase:

DNA polymerase is the chief enzyme of DNA replication. DNA polymerase activity was discovered by Arthur Kornberg in

1956. E. coli has four more enzymes, DNA polymerase II, III (Table. 28.1), IV and V; DNA polymerase III (Pol III) is concerned with DNA replication, while the remaining four enzymes are involved in DNA repair.

All DNA polymerases require a template strand, which is copied. DNA polymerases also require a primer, which is complementary to the template. The reaction of DNA

polymerases is thus better understood as the addition of nucleotides to a primer to make a sequence complementary to a template. The requirement for template and primer are exactly what would be expected of a replication enzyme. Because DNA is the information store of the cell, any ability of DNA polymerases to make DNA sequences from nothing would lead to the degradation of the cell's information copy.

All DNA polymerases require the following:

(1) A template DNA strand, (2) A short primer (either RNA or DNA), and (3) A free 3′ -OH in the primer.

They add one nucleotide at a time to the free 3′ -OH of the primer, and extend the primer chain in 5′ → 3 ′ direction.

• More than one DNA polymerase exists in each cell.

Figure 1 : Arthur Kornberg

single polypeptides, but DNA polymerase III is a ten subunits protein with a molecular mass of approx. 900KD.

• DNA polymerase III enzyme is responsible for DNA replication in vivo. It has 5’→ 3 ′ polymerase and 3’→ 5 ′ exonuclease activities. It catalyzes DNA synthesis at very high rates, e.g., 15,000 bases/min at 37°C. It is composed of several subunits.
• In case of eukaryotes , at least nine different DNA polymerases are found; Table 28.2 lists the properties of five of these enzymes. DNA polymerase δ replicates the leading strand, while DNA polymerase ϵ synthesizes the lagging strand.

DNA polymerase α catalyzes priming of both the strands. DNA polymerases ξ, η, τ, and k are all nuclear DNA repair enzymes. DNA polymerase γ is found in

mitochondria and catalyzes replication of mtDNA.

Enzyme # Endonucleases:

An endonuclease produces an internal cut (single- or double-stranded) in a DNA

molecule. But a restriction endonuclease produces cuts only at those sites that have a

specific base sequence. During DNA replication, an endonuclease may induce a nick to

initiate DNA replication, or it may induce nicks to generate a swivel for DNA unwinding.

Enzyme # DNA gyrase

DNA gyrase, or simply gyrase, is an enzyme within the class of topoisomerase (Type II

topoisomerase) that relieves strain while double-stranded DNA is being unwound by

helicase. This causes negative supercoiling of the DNA. The gyrase supercoils (or relaxes

positive supercoils) into DNA by looping the template so as to form a crossing, then

cutting one of the double helices and passing the other through it before releasing the

break, changing the linking number by two in each enzymatic step.

Enzyme # Helicase:

Helicase effects strand separation at the forks and use one ATP molecule for each base that is separated. In E. coli, DNA functions as helicase; this protein is a hexamer and it moves with the replication fork. Helicase breaks the hydrogen bonding between bases.

Enzyme # Single-Strand Binding (SSB) Protein:

SSB protein binds to single-stranded DNA, and prevents it from forming duplex DNA or

secondary structures. SSB binds as a monomer, but it binds cooperatively in that binding

of one SSB molecule facilitates binding of more SSB monomers to the same DNA strand.

E. coli SSB is a tetramer.

Enzyme # Polynucleotide Ligase:

DNA ligase or polynucleotide ligase catalyzes the formation of phosphodiester linkage between two immediate neighbour nucleotides of a DNA strand. Thus it seals the nicks remaining in a DNA strand either following DNA replication or DNA repair. However, this enzyme cannot fill the gaps in DNA strands.

Figure: Stepwise action of enzymes involved in the replication

References

• https://medicalbiochemistry142.wordpress.com/2014/04/04/clinical-significance-of- topoisomerase-inhibitors/
• http://www.biologydiscussion.com/cell/prokaryotes/enzymes-involved-in-dna-replication- prokaryotes/