φ29 DNA polymerase

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φ29 DNA polymerase
φ29 DNA polymerase
according to PDB  1XHX
other names

Bacillus phage φ29 gene product 2

Existing structure data : PDB  1XHZ , PDB  1XI1

Mass / length primary structure 575 amino acids , 66,714 Da
Identifier
External IDs
Enzyme classification
EC, category 2.7.7.7
Orthologue (Bacillus phage φ29)
Entrez 6446511
UniProt P03680


PubMed search 6446511

The φ29 DNA polymerase is an enzyme from the group of DNA polymerases and is produced by the φ29 bacteriophage .

properties

As a type B DNA polymerase, it extends DNA sequences at the 3 'end if they occur as a DNA double strand and at least one single strand is present as a template after the 3' end . It is used by the phage to replicate DNA in the viral genome . In the case of circular DNA such as plasmids , rolling circle replication takes place . During replication, the φ29 DNA polymerase forms a heterodimer with the primer terminal protein (TP) and binds to the origin of replication at one of the two 5 'ends of the phage DNA. The hydroxy group of serine at position 232 serves as a primer . φ29 DNA polymerase has three enzymatic activities : DNA replication, Desoxynukleotidylierung of TP with deoxy adenosine monophosphate via an organophosphate-transition state and a 3 '→ 5' - exonuclease type II error correction ( proof-reading ). While the exonuclease ensures a low error rate during synthesis, the binding of the TP and the beginning of the synthesis are comparatively incorrect. It starts at the second thymine base and then slides back a few bases. The φ29 DNA polymerase is strand-shifting, which means that synthesis can also take place on a double strand. The leucine at position 384 is involved in the specificity of binding of deoxynucleotides . Tyrosines at positions 226 and 390 are involved in translocation along the template. The Φ29 DNA polymerase has a higher affinity for single-stranded than for double-stranded DNA. The tyrosines at positions 256 and 390 increase the affinity of nucleotide binding. The tyrosine at position 59, the histidine at position 61 and a phenylalanine at position 69 serve to bind the exonuclease to DNA. The φ29 DNA polymerase is not a thermostable DNA polymerase .

The Φ29 DNA polymerase catalyzes the reaction:

Deoxynucleoside triphosphate + DNA (n) diphosphate + DNA (n + 1)

Applications

The φ29 DNA polymerase is used in biochemistry for isothermal DNA amplification , e.g. B. the Gibson Assembly . A thermostable alternative is thermostable at 65 ° C large fragment of Bst DNA polymerase from Bacillus stearothermophilus .

The Φ29 DNA polymerase is used to copy from genomes used ( Genomamplifikation , engl. Whole genome amplification , WGA), since they have a high processivity , a low error rate (for proof reading ), a low bias owns and because long DNA fragments over 10kb and a higher product concentration than thermostable DNA polymerases . As an isothermal method, no thermal cycler is required; random nucleotide hexamers are often used as primers in WGA.

literature

Web links

Individual evidence

  1. ↑ FB Dean, JR Nelson, TL Giesler, RS Lasken: Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. In: Genome Research. Volume 11, number 6, June 2001, pp. 1095-1099, doi : 10.1101 / gr.180501 , PMID 11381035 , PMC 311129 (free full text).
  2. ↑ V. Truniger, JM Lázaro, M. de Vega, L. Blanco, M. Salas: phi 29 DNA polymerase residue Leu384, highly conserved in motif B of eukaryotic type DNA replicases, is involved in nucleotide insertion fidelity. In: The Journal of Biological Chemistry. Volume 278, Number 35, August 2003, pp. 33482-33491, doi : 10.1074 / jbc.M303052200 , PMID 12805385 .
  3. ↑ JM Dahl, H. Wang, JM Lázaro, M. Salas, KR Lieberman: Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi} 29 DNA polymerase. In: The Journal of Biological Chemistry. Volume 289, number 10, March 2014, pp. 6350-6361, doi : 10.1074 / jbc.M113.535666 , PMID 24464581 , PMC 3945302 (free full text).
  4. ^ J. Saturno, L. Blanco, M. Salas, JA Esteban: A novel kinetic analysis to calculate nucleotide affinity of proofreading DNA polymerases. Application to phi 29 DNA polymerase fidelity mutants. In: The Journal of Biological Chemistry. Volume 270, Number 52, December 1995, pp. 31235-31243, PMID 8537389 .
  5. ↑ M. de Vega, JM Lázaro, M. Salas: Phage phi 29 DNA polymerase residues involved in the proper stabilization of the primer terminus at the 3'-5 'exonuclease active site. In: Journal of Molecular Biology. Volume 304, Number 1, November 2000, pp. 1-9, doi : 10.1006 / jmbi.2000.4178 , PMID 11071805 .
  6. ↑ Alsmadi O, Alkayal F, Monies D, Meyer BF: Specific and complete human genome amplification with improved yield achieved by phi29 DNA polymerase and a novel primer at elevated temperature . In: BMC Res Notes . 2, 2009, p. 48. doi : 10.1186 / 1756-0500-2-48 . PMID 19309528 . PMC 2663774 (free full text).
  7. ↑ L. Blanco, A. Bernad, JM Lázaro, G. Martín, C. Garmendia, M. Salas: Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. In: The Journal of biological chemistry. Volume 264, Number 15, May 1989, pp. 8935-8940, PMID 2498321 .
  8. ↑ Pugh TJ, Delaney AD, Farnoud N, etal: Impact of whole genome amplification on analysis of copy number variants . In: Nucleic Acids Res . 36, No. 13, August 2008, p. E80. doi : 10.1093 / nar / gkn378 . PMID 18559357 . PMC 2490749 (free full text).
  9. ↑ a b Pinard R, de Winter A, Sarkis GJ, etal: Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing . In: BMC Genomics . 7, 2006, p. 216. doi : 10.1186 / 1471-2164-7-216 . PMID 16928277 . PMC 1560136 (free full text).