Other DNA polymerases:
DNA-Pol of B.subtilis:
DNA Pol from Thermophilic Bacteria:
DNA POL -Solfobolus. Acidocaldarius; http://www.rcsb.org
Thermus aquaticus DNA- Polymerase:
DNA polymerase;Thermophilus aquaticus; www.hindawi.com
T7 DNA polymerase:
· The polymerase is product of phages gp5 gene.
· The protein is a single polypeptide chain of Mol.wt 79.7 KD.
· In host cells, it is complexed with hosts accessory subunit of 11.7 KD called thioredoxins; they are associated in 1:1 ratio.
· Many phages like F2, QB and ms2 and other RNA phages are also associated with host components.
· Structurally the T7 DNA polymerase has similarities with Large Fragment (Klenow) of E.coli DNA pol-I and alpha subunit of DNA pol-III.
· The T 7 polymerase with theoredoxin exhibits high processivity and high fidelity.
· The gp5 gene product perse shows 3ΰ5 exonuclease activity on ssDNA as well as on dsDNA.
· With thioredoxins it exhibits full polymerase activity.
· This enzyme lacks 5 exonuclease activity.
· The gp6 gene is located downstream of the gp5.
· T he gp6 protein shows 5->3 exonuclease activity.
T7DNA pol/Trx/DNA; Doublie, Tabor, Long, Richardson & Ellenberger, Nature 391:251-258, 1998www.personal.psu.edu
· Structurally it shows strong homology with the N-terminal segment of DNA pol-I.
· The combined mass of gp 5 and gp6 is almost same as that of DNA pol I.
· The combination of gp5, gp6 and thioredoxin has 5-3 polymerase, 53 exonuclease and 35 exonuclease activity.
· It is a unique combination of features, which suggest, how recombination of independent gene segments, during evolution, can give rise to genes whose products have multiple activities.
· The reverse is also true. It means that a single gene with different structural and functional coding domains can be recombined in different ways to be distributed to different genes or they work as independent genes. These are some of the steps or modes by which natural selection and evolution operates at molecular level.
· Another gene product, gp 4 has very unique functions; it has helicase as well as primase activities.
· This protein produces just a tetra nucleotide RNA primer.
· The T7 DNA polymerase, without EDTA gets modified during purification process.
· This leads to loss of its 3ΰ5 exonuclease activity. This loss is due to the selective oxidation and modification of an amino acid residue in the vicinity of iron binding site in the protein.
· Iron ion is very essential for 3->5 exonuclease activity.
· Commercially available enzymes lack 35 exonuclease activity, and they dont discriminate dNTPs from normal ddNTPS (di Deoxy NTPs). The enzyme even tolerates d ITPs in the place of dGTPs.
· Substituting Mg2+ with Mn 2+ further enhances the lack of discrimination between dNTPs from that of dideoxy NTPs.
· Because of the above features T7 DNA polymerase is extensively used in DNA sequencing experiments.
· It is also used in a variety of genetic engineering techniques.
· Presently this enzyme is available as genetically modified product and sold by by many commercial Biotechnology firms as Sequenase.
· In E. coli, DNA polymerase IV (Pol 4) is an error-prone DNA polymerase involved in non-targeted mutagenesis. Pol IV is a Family Y polymerase expressed by the dinB gene that is switched on via SOS induction caused by stalled polymerases at the replication fork. During SOS induction, Pol IV production is increased 10-fold 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. Another function of Pol IV is to perform tanslesion synthesis at the stalled replication fork like, for example, bypassing N2-deoxyguanine adducts at a faster rate than traversing undamaged DNA. Cells lacking dinB gene have a higher rate of mutagenesis caused by DNA damaging agents. http://en.wikipedia.org/wiki/DNA_polymerase
DNA polymerase V (Pol V) is a Y-family DNA polymerase that is involved in SOS response and translesion synthesis DNA repair mechanisms. Transcription of Pol V via the umuDC genes is highly regulated to only produce Pol V when damaged DNA is present in the cell generating an SOS response. Stalled polymerases cause RecA to bind to the ssDNA which causes the LexA protein to autodigest. LexA then loses is ability to repress the transcription of the umuDC operon. The same RecA-ssDNA nucleoprotein posttranslationally modifies the UmuD protein into UmuD' protein. UmuD and UmuD' form a heterodimer that interacts with UmuC which in turn activates umuC's polymerase catalytic activity on damaged DNA. http://en.wikipedia.org/wiki/DNA_polymerase
Motifs and homologies among polymerases:
Amino acid sequences of certain segments of polymerases show significant homologies among several DNA-Pols, RNA dependent DNA- polymerases, DNA and RNA dependent RNA-polymerases. The active sites, where the template bind and nucleotides bind have more or less conserved in both DNA and RNA polymerases, because they have same mode of action and perform similar functions.