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Ab initio Synthesis by DNA Polymerases

Ab Initio Synthesis by DNA Polymerases

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Ab initio Synthesis by DNA Polymerases

  1. 1. Authors Nadezhda V. Zyrina, Valeriya N. Antipova & Lyudmila A. Zheleznaya Presented by: Amna Jalil
  2. 2. Introduction The polymerization of free nucleotides into new genetic elements by DNA polymerases in the absence of DNA, called ab initio DNA synthesis little known phenomenon
  3. 3. In general, template-independent generation of genetic information by DNA polymerases is a known process. A number of error-prone DNA polymerases efficiently incorporate nucleotides in DNA lesions where template information is absent.
  4. 4. Another instance of template-independent nucleotide polymerization is terminal deoxynucleotidyl transferase like activity, achieved by adding dNTPs to the 3′-OH terminus of a blunt-ended duplex DNA substrate.
  5. 5. Ab initio synthesis takes place in the absence of any added DNA. Ab initio DNA synthesis, has been known for 50 years, incontrovertible evidence was only obtained in the last decade.
  6. 6. History of ab initio DNA synthesis research In a typical replication mode, initiation of DNA synthesis requires:  A template DNA strand and  A primer  A short oligonucleotide complementary to the template DNA region with a free 3′- OH terminus
  7. 7. • In the 1960 and 1970s it was shown that some prokaryotic DNA polymerases are capable of providing the de novo synthesis of poly (dA-dT) and poly (dG) poly (dC) without any added primer or template DNA.
  8. 8. These studies were conducted with partially purified preparations of enzymes; the scientific community assumed that this synthesis might be due to contamination by DNA or other enzymes and these data have therefore not been given due attention.
  9. 9. 30 years later was it convincingly demonstrated that highly purified thermophilic DNA polymerases Tli and Tth were able to synthesize about 50 kb of DNA without any template and primer. This phenomenon was called ‘creative’, or ab initio DNA synthesis.
  10. 10. The possibility of DNA contamination in the reaction mixture, which may serve as a primer and/or template, was vigorously excluded. The synthesized double-stranded DNAs had mainly:  Short repetitive and palindromic sequences  GC content was about 25% The reaction conditions (temperature, ionic strength, and pH) were extremely important for this reaction
  11. 11. Based on these findings, Ogata & Miura suggested that genetic information might be created directly by protein.
  12. 12. The primer/template-independent polymerization appeared to proceed via two reactions  Slow formation of 16–19-nt-long oligo(dA-T) without primer/template Rapid elongation of the oligo(d A-T) by self-priming
  13. 13. Ab initio synthesis in the presence of restriction endonucleases Ab initio DNA synthesis was extremely enhanced if a thermostable restriction endonuclease (Tsp509I, TspRI, etc.) was added to the reaction with thermophilic DNA polymerase (Vent, Bst and 9ºNm).
  14. 14. ◊ The high efficiency of this synthesis resulted from the exponential amplification involving digestion /elongation cycles. ◊ A longer DNA with numerous recognition sites was digested to short fragments, and the short fragments were used as seeds for elongation to synthesize longer DNA.
  15. 15. Ab initio synthesis stimulated by nicking endonucleases Very intensive ab initio synthesis takes place in the presence of nicking endonuclease Nt.BspD6I Similar to restriction endonucleases, nicking endonucleases recognize a short specific sequence in double stranded DNA and cleave DNA at a fixed position relative to the recognized sequence.
  16. 16. However, unlike restriction endonucleases, nicking endonucleases make a nick in only one, predetermined DNA strand.
  17. 17. • The macromolecular structure and the characteristics of the sequence DNAs synthesized in the presence of Nt.BspD6I differed from those synthesized by DNA polymerases alone or in the presence of restriction endonucleases. • Some of DNA molecules had a branched structure.
  18. 18. The sequences of DNA were represented mainly by non-palindromic containing Nt.BspD6I recognition site (GAGTC)
  19. 19. Ab initio synthesis stimulated by DnaB helicase New DNA molecules over 100 kbp long can be synthesized without preexisting matrices when helicase DnaB is added to a reaction mixture with: Thermophilic polymerases Mesophilic polymerases
  20. 20. The synthesized double-stranded DNA had: Single-stranded stretches A–T-rich sequence Highly repetitive sequence
  21. 21. A hypothetical model of ab initio DNA synthesis by DNA polymerases Initiation of ab initio DNA synthesis remains a mystery. The hypothetical mechanism of ab initio DNA synthesis can be described with several stages.
  22. 22. In the initial step, DNA polymerase generates a pool of oligonucleotides with random sequences. At the following stage, oligonucleotides with specific sequences, which can ‘facilitate’ their own replication, are amplified. Palindromic sequences are preferable because they can form reversible hairpin structures at their 3′-termini, thus priming the DNA elongation
  23. 23. Very long DNA stretches may be synthesized through multiple strand displacement reactions on the 3′-termini of formed hairpins A restriction endonuclease may somehow help DNA polymerase to select the sequence to be synthesized.
  24. 24. However, these models do not entirely explain how non-palindromic repeats propagate. The formation of hairpins in non palindromic sequences is not possible either in the middle or at the ends of the molecule.
  25. 25. A possible functional role of ab initio DNA synthesis Tandem sequences consisting of short repeats occur in all genomes. A comparison of ab initio DNA sequences with those of the known natural DNAs revealed that very similar tandem repeats are present in coding and non-coding regions. This fact suggests that repeating sequences were synthesized by DNA polymerase in a template-independent manner.
  26. 26. Conclusions The major problem of numerous nucleic acid amplification methods is the accumulation of non-specific products, which hamper identification of specific sequences. This process may be a result of ab initio DNA synthesisby thermophilic DNA polymerases.
  27. 27. Besides PCR, some other nucleic acid amplification techniques Strand displacement amplification Rolling circle amplification Exponential amplification reaction also hampered by non-specific synthesis
  28. 28.  However, the problem of nonspecific amplification still remains unsolved.  A new strategy was offered for isothermal DNA amplification in the presence of nicking enzymes .  It was based on the use of SSB proteins as inhibitors of non-specific ab initio synthesis.  One of the proteins, T4 gp32, almost completely inhibited ab initio DNA synthesis.
  29. 29. The knowledge gained will increase our understanding of how DNA polymerases function and will also suggest future research in molecular biology. The results may be very useful to develop techniques requiring fast and inexpensive preparation of large amounts of DNA.