Enzymes used in DNA Replication This document holds the enzymes used in DNA replication, their pictorial

representation and functioning.

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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

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• The key distinction among the enzyme forms is their processitivity—how long a chain they synthesize before falling off the template. A DNA polymerase used in replication is more processive than a repair enzyme. The replication enzyme needs to make a long enough chain to replicate the entire chromosome. The repair enzyme needs only to make a long enough strand to replace the damaged sequences in the chromosome.

• What if polymerase incorporates a wrong base in the growing chain, suppose adenine has to be incorporated in the chain and guanine comes and get incorporated in the chain, in this case this incorporation a mutation which might lead to some serious adverse effect on the future generation, to rectify these errors DNA polymerase is known to scrutinize the new bases added to the growing chain and to delete or remove the wrong bases; this is called proof-reading. Proof-reading activity reduces errors in replication by over 100 – fold.

• The best-studied bacterium, E. coli, has three DNA polymerase types.

A. DNA Polymerase I:

• DNA polymerase I is simplest and best understood of the DNA polymerases, not responsible for most chromosomal DNA replication in E. coli, but functions in vivo both in DNA replication and DNA repair.

This enzyme has the following three activities:

(1) The 5′ → 3′ polymerase activity is responsible for primer extension or DNA synthesis.

(2) The 5′ → 3′ exonuclease activity is involved in excision of DNA strands during DNA repair; it removes ~ 10 bases at a time. An exonuclease digests nucleic acids (here DNA) from one end, and it does not cut DNA internally.

(3) The 3′ → 5′ exonuclease activity is responsible for proof-reading.

DNA polymerase I is encoded by gene polA, has a single polypeptide, and can initiate replication in vitro at a nick in a DNA duplex. It can be cleaved by proteolytic treatments into a large and a small fragments. This large fragment, called Klenow fragment, lacks 5′ → 3′ exonuclease activity and is used for in vitro DNA replication.

B. DNA Polymerase II:

• DNA polymerase II enzyme functions in DNA-repair. It has 5′ → 3′ polymerase and 3′ → 5′ exonuclease activities, and uses as template only such DNA duplexes that have short gaps.

C. DNA Polymerase III:

• Now we know that DNA polymerase III, isolated in 1972, is involved in replication along with DNA polymerase I. DNA polymerase I and II are

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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.

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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.

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Figure: Working of helicase, SSB proteins and gyrase.

Enzyme # Primase:

This enzyme activity catalyzes the synthesis of RNA primers to initiate DNA replication. In E. coli, DnaG functions as primase. But in eukaryotes, DNA polymerase α provides this function. There are, however, several other ways in which primers are produced, e.g., the 3′-OH generated by a nick in the template DNA molecule.

Figure: it shows the cumulative function of single strand proteins and primers

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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.

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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/55416