DNA POLYMERASES

DNA polymerases are enzymes that catalyze

the template-directed synthesis of DNA.

DNA dependent DNA polymerase

Catalyzes DNA template directed extension of the 3’-end of a DNA strand .

Cannot initiate a chain de novo.

Requires a primerwhich may be DNA or RNA.

RNA dependent DNA polymerase

Reverse transcriptase

Catalyzes RNA template directed extension of the 3’-end of a DNA strand .

Cannot initiate a chain de novo.

Requires a primerwhich may be DNA or RNA.

Discovery and history

Discovered DNA

polymerase in E.coli in

1955.

Won noble prize for the

discovery.

Discovery of DNA

polymerase I.

Arthur

Kornberg

DNA polymerases family

A - includes the archetypal E. coli DNA polymerase I.

B - includes all the eukaryotic polymerases involved in

chromosome replication.

RT

X Include enzymes for DNA repair or specialized

Y types of replication.

Family

PROKARYOTES EUKARYOTES

DNA polymerase I

DNA polymerase II

DNA polymerase III

DNA polymerases IV

DNA polymerase V

More than 15

DNA polymerases α,

δ, and ϵ

DNA polymerase γ

DNA polymerases in ProkaryotesDNA polymerase I

This is a repair polymerase and is involved in excision repair with 3'-5' and 5'-3'exonuclease activity and processing of Okazaki fragments generated during laggingstrand synthesis.

Most abundant polymerase accounting for >95% of polymerase activity in E. coli.

Cells lacking Pol I have been found suggesting Pol I activity can be replaced by theother four polymerases.

Pol I adds ~15-20 nucleotides per second.

DNA polymerase II Pol II has 3'-5' exonuclease activity and participates in DNA repair.

Pol II is also thought to be a backup to Pol III as it can interact with holoenzyme proteins and assume a high level of processivity.

The main role of Pol II is thought to be the ability to direct polymerase activity at the replication fork and help stalled Pol III bypass terminal mismatches.

DNA polymerase III Primary enzyme involved in DNA replication in E. coli and belongs to Family C

polymerases.

The core consists of three subunits - α, the polymerase activity hub, ɛ,

exonucleolytic proofreader, and θ, which may act as a stabilizer for ɛ.

The holoenzyme contains two cores, one for each strand, the lagging and

leading.

The beta sliding clamp processivity factor is also present in duplicate, one for

each core, to create a clamp that encloses DNA allowing for high processivity.

DNA polymerase IVAn error-prone DNA polymerase involved in non-targeted mutagenesis.

During SOS induction, Pol IV production is increased tenfold and one of the

functions during this time is to interfere with Pol III holoenzyme processivity.

This creates a checkpoint, stops replication, and allows time to repair DNA lesions

via the appropriate repair pathway.

DNA polymerase V

Pol V is a Y-family DNA polymerase that is involved

in SOS response and translesion synthesis DNA repair

mechanisms.

DNA polymerases in Eukaryotes

Semi-conservative replication of DNA.

Use of single stranded DNA chain as a template and

four deoxynucleotides(TTP, dCTP, dGTP, dATP)as

precursors for DNA synthesis.

Assembly of the precursor nucleotides on the template

to form a complementary DNA strand, selecting the

incoming nucleotide using the base pair rules A.T and

G.C.

To start synthesis on a single stranded DNA molecule,

DNA polymerases need a primer.

Primer : length of RNA or DNA that is annealed to the

single-stranded template.

Polymerases Involved in

Chromosome Replication

polymerase a – has an associated primase activity

capable of synthesizing short (10 nucleotide)RNA

primers.

DNA polymerase a is the only enzyme that could be

involved in the primer synthesis during initiation at

origins of replication.

a polymerase is also required during the elongation

step for the priming of synthesis of Okazaki

fragments on the lagging strand.

After DNA polymerase a has synthesized a short (30–40 nucleotide)stretch of DNA, a process called polymerase switching takes place in which polymerase a is displaced from the template and synthesis by polymerases d and probably e takes over.

Polymerase d is a multi subunit polymerase and probably functions at the leading and lagging strands of the replication fork.

Polymerase e consists of four subunits, and its precise role in chromosomal replication is unclear.

It has been proposed that the function of DNA polymerase e may be restricted to the lagging strand, perhaps only in the maturation of Okazaki fragments.

Polymerases Involved in DNA

Repair

Polymerase b - In base-excision repair, removal of an altered base is followed by excision of a single abasicnucleotide. DNA polymerase b then serves to fill in the missing nucleotide; this involves an activity in the N-terminal domain of the protein that removes the 5’ phosphate remaining after nucleotide excision, followed by a polymerization reaction to fill in the missing nucleotide.

Polymerases a, b, g - in conjunction with the RFC and

PCNA as cofactors.

3D structure of the DNA-

binding helix-turn-helix motifs in

human DNA polymerase beta

Polymerases with Specialized Functions

Polymerase s - has recently been discovered to be necessary for ensuring that the two replicated chromosomes (sister chromatids) remain attached together after DNA replication. This continued association is important for ensuring the proper separation of the chromosomes in mitosis (Wang et al., 2000).

Telomerase - required to complete chromosome synthesis, for replication of the telomeres at the ends of chromosomes. This enzyme is actually an RNA-directed DNA polymerase, and is unusual in that it is not template directed but uses an internal RNA molecule to direct synthesis of short repeated sequences that are added to the ends of chromosomes.

Polymerase g- found in mitochondria and is required for replication and repair of the mitochondrial DNA.

APPLICATIONS

DNA cloning

The polymerase chain reaction (PCR)

DNA sequencing

Single nucleotide polymorphism (SNP) detection

Whole genome amplification (WGA)

Synthetic biology

Molecular diagnostics.

The polymerase chain reaction (PCR)

DNA polymerase I from Thermus aquaticus (Taqpolymerase) is widely used in PCR.

Isolated in 1976 from hot springs in Yellowstone National Park), where it thrives at 70° C.

Can be activated by heating the sample and remains active with the high temperatures required to denature DNA strands (typically 94°C).

Allows repeated cycles of denaturing, annealing and extension (thermo cycling) without the need to add additional polymerase at each cycle.

Pyrococcus furiosus (Pfu) Hyperthermophilic archaeon.

Discovered in the Lower Geyser Basin of Yellowstone National Park.

Its DNA polymerase (Pfu) has been used in many PCR applications.

Pyrococcus furiosus has a feature that is absent in Taqpolymerase.

An exonuclease domain that has 3′–5′ exonucleaseactivity.

This allows Pfu to proofread using a conformational change .

DNA sequencing technologies

Sanger DNA sequencing was used to sequence

the first draft of the human genome in and remains

a standard and widespread method to determine

DNA sequence.

New next generation sequencing methods have

dramatically increased sequencing output while

lowering costs.

DNA polymerases have been engineered to

incorporate the modified nucleotides used in DNA

sequencing, genotyping, and synthesis of artificial

DNA.

Molecular diagnostics Isothermal amplification techniques such as Loop-

Mediated Amplification (LAMP) have been routinely

used as diagnostic tests to detect infectious disease.

An engineered thermo-stable viral polymerase

with RT and DNA polymerase activities that can

be used in isothermal RT-LAMP detection of RNA

has been described.

What’s in the future ? Molecular biology will move toward analysis of low

concentration bio molecules (i.e., a single set of chromosomes).

Novel amplification techniques are also required to profile genetic variations among single cells because the quantity of genomic DNA from a single cell is insufficient to sequence directly. Therefore, DNA must first be amplified prior to further analysis.

Synthetic biology aims to design new biological systems such as genetic pathways, operons, and genomes and thus may require long, chromosome-size, amplification.

Current DNA polymerases introduce errors during amplification and thus DNA polymerases with very low error rates are needed to ensure that long, amplified DNA are exact copies of the starting material.