Prokaryotic DNA replication

Prokaryotic DNA replication is the process by which a prokaryote duplicates its entire genome into another copy that is passed on to daughter cells.[1] Although it is often studied in the model organism E. coli, other bacteria show many similarities.[2] Replication is bi-directional and originates at a single origin of replication (OriC).[3] It consists of three steps: Initiation, elongation, and termination.[4]

Most circular bacterial chromosomes are replicated bidirectionally, starting at one point of origin and replicating in two directions away from the origin. This results in semiconservative replication, in which each new identical DNA molecule contains one template strand from the original molecule, shown as the solid lines, and one new strand, shown as the dotted lines.

Initiation

DNA replication begins at the origin of replication, a region commonly containing repeating sequences (DnaA boxes) that bind DnaA, an initiation protein.[5] DnaA-ATP will first bind high-affinity boxes (R1, R2, and R4, which have a highly conserved 9 bp consensus sequence 5' - TTATCCACA - 3'[2]), then oligomerize into several low-affinity boxes.[6] This accumulation will displace a protein called Fis, allowing another protein, IHF, to bind the DNA and induce a bend.[6] This bend allows the DnaA chain to load onto an AT-rich region of 13-mers (the DUE, Duplex unwinding element), causing the double-stranded DNA to separate.[2] The DnaC helicase loader will interact with the DnaA on the single-stranded DNA to recruit the DnaB helicase,[7] which will continue to unwind the DNA as the DnaG primase lays down an RNA primer and DNA Polymerase III holoenzyme begins elongation.[8]

Regulation

Chromosome replication in bacteria is regulated at the initiation stage.[2] DnaA-ATP is hydrolyzed into the inactive DnaA-ADP by RIDA (Regulatory Inactivation of DnaA),[9] and converted back to the active DnaA-ATP form by DARS (DnaA Reactivating Sequence, which is itself regulated by Fis and IHF).[10][11] However, the main source of DnaA-ATP is synthesis of new molecules.[2] Meanwhile, several other proteins interact directly with the oriC sequence to regulate initiation, usually by inhibition. In E. coli these proteins include DiaA,[12] SeqA,[13] IciA,[2] HU,[7] and ArcA-P,[2] but they vary across other bacterial species. A few other mechanisms in E. coli that variously regulate initiation are DDAH (datA-Dependent DnaA Hydrolysis, which is also regulated by IHF),[14] inhibition of the dnaA gene (by the SeqA protein),[2] and reactivation of DnaA by the lipid membrane.[15]

Elongation

Once priming is complete, DNA polymerase III holoenzyme is loaded into the DNA and replication begins. The catalytic mechanism of DNA polymerase III involves the use of two metal ions in the active site, and a region in the active site that can discriminate between deoxyribonucleotides and ribonucleotides. The metal ions are general divalent cations that help the 3' OH initiate a nucleophilic attack onto the alpha phosphate of the deoxyribonucleotide and orient and stabilize the negatively charged triphosphate on the deoxyribonucleotide. Nucleophilic attack by the 3' OH on the alpha phosphate releases pyrophosphate, which is then subsequently hydrolyzed (by inorganic phosphatase) into two phosphates. This hydrolysis drives DNA synthesis to completion.

Furthermore, DNA polymerase III must be able to distinguish between correctly paired bases and incorrectly paired bases. This is accomplished by distinguishing Watson-Crick base pairs through the use of an active site pocket that is complementary in shape to the structure of correctly paired nucleotides. This pocket has a tyrosine residue that is able to form van der Waals interactions with the correctly paired nucleotide. In addition, dsDNA (double stranded DNA) in the active site has a wider major groove and shallower minor groove that permits the formation of hydrogen bonds with the third nitrogen of purine bases and the second oxygen of pyrimidine bases. Finally, the active site makes extensive hydrogen bonds with the DNA backbone. These interactions result in the DNA polymerase III closing around a correctly paired base. If a base is inserted and incorrectly paired, these interactions could not occur due to disruptions in hydrogen bonding and van der Waals interactions.

DNA is read in the 3' → 5' direction, therefore, nucleotides are synthesized (or attached to the template strand) in the 5' → 3' direction. However, one of the parent strands of DNA is 3' → 5' while the other is 5' → 3'. To solve this, replication occurs in opposite directions. Heading towards the replication fork, the leading strand is synthesized in a continuous fashion, only requiring one primer. On the other hand, the lagging strand, heading away from the replication fork, is synthesized in a series of short fragments known as Okazaki fragments, consequently requiring many primers. The RNA primers of Okazaki fragments are subsequently degraded by RNase H and DNA Polymerase I (exonuclease), and the gaps (or nicks) are filled with deoxyribonucleotides and sealed by the enzyme ligase.

Rate of replication

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli.[16] During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.[17]

Termination

Termination of DNA replication in E. coli is completed through the use of termination sequences and the Tus protein. These sequences allow the two replication forks to pass through in only one direction, but not the other.

DNA replication initially produces two catenated or linked circular DNA duplexes, each comprising one parental strand and one newly synthesised strand (by nature of semiconservative replication). This catenation can be visualised as two interlinked rings which cannot be separated. Topoisomerase 2 in E. coli unlinks or decatenates the two circular DNA duplexes.

References

  1. ↑ "What is DNA Replication?". yourgenome.org. Wellcome Genome Campus. Retrieved 24 February 2017.
  2. 1 2 3 4 5 6 7 8 Wolański M, Donczew R, Zawilak-Pawlik A, Zakrzewska-Czerwińska J (2014-01-01). "oriC-encoded instructions for the initiation of bacterial chromosome replication". Frontiers in Microbiology. 5: 735. PMC 4285127 Freely accessible. PMID 25610430. doi:10.3389/fmicb.2014.00735.
  3. ↑ Bird RE, Louarn J, Martuscelli J, Caro L (October 1972). "Origin and sequence of chromosome replication in Escherichia coli". Journal of Molecular Biology. 70 (3): 549–66. PMID 4563262.
  4. ↑ Bussiere DE, Bastia D (March 1999). "Termination of DNA replication of bacterial and plasmid chromosomes". Molecular Microbiology. 31 (6): 1611–8. PMID 10209736. doi:10.1046/j.1365-2958.1999.01287.x.
  5. ↑ Rajewska M, Wegrzyn K, Konieczny I (March 2012). "AT-rich region and repeated sequences - the essential elements of replication origins of bacterial replicons". FEMS Microbiology Reviews. 36 (2): 408–34. PMID 22092310. doi:10.1111/j.1574-6976.2011.00300.x.
  6. 1 2 Riber L, Frimodt-Møller J, Charbon G, Løbner-Olesen A (2016-01-01). "Multiple DNA Binding Proteins Contribute to Timing of Chromosome Replication in E. coli". Frontiers in Molecular Biosciences. 3: 29. PMC 4924351 Freely accessible. PMID 27446932. doi:10.3389/fmolb.2016.00029.
  7. 1 2 Kaguni JM (October 2011). "Replication initiation at the Escherichia coli chromosomal origin". Current Opinion in Chemical Biology. 15 (5): 606–13. PMC 3189269 Freely accessible. PMID 21856207. doi:10.1016/j.cbpa.2011.07.016.
  8. ↑ Ozaki S, Noguchi Y, Hayashi Y, Miyazaki E, Katayama T (October 2012). "Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex". The Journal of Biological Chemistry. 287 (44): 37458–71. PMC 3481341 Freely accessible. PMID 22942281. doi:10.1074/jbc.M112.372052.
  9. ↑ Kato J, Katayama T (August 2001). "Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli". The EMBO Journal. 20 (15): 4253–62. PMC 149159 Freely accessible. PMID 11483528. doi:10.1093/emboj/20.15.4253.
  10. ↑ Fujimitsu K, Senriuchi T, Katayama T (May 2009). "Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA". Genes & Development. 23 (10): 1221–33. PMC 2685538 Freely accessible. PMID 19401329. doi:10.1101/gad.1775809.
  11. ↑ Kasho K, Fujimitsu K, Matoba T, Oshima T, Katayama T (December 2014). "Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation". Nucleic Acids Research. 42 (21): 13134–49. PMC 4245941 Freely accessible. PMID 25378325. doi:10.1093/nar/gku1051.
  12. ↑ Ishida T, Akimitsu N, Kashioka T, Hatano M, Kubota T, Ogata Y, Sekimizu K, Katayama T (October 2004). "DiaA, a novel DnaA-binding protein, ensures the timely initiation of Escherichia coli chromosome replication". The Journal of Biological Chemistry. 279 (44): 45546–55. PMID 15326179. doi:10.1074/jbc.M402762200.
  13. ↑ Frimodt-Møller J, Charbon G, Løbner-Olesen A (December 2016). "Control of bacterial chromosome replication by non-coding regions outside the origin". Current Genetics. PMID 27942832. doi:10.1007/s00294-016-0671-6.
  14. ↑ Kasho K, Katayama T (January 2013). "DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation". Proceedings of the National Academy of Sciences of the United States of America. 110 (3): 936–41. PMC 3549119 Freely accessible. PMID 23277577. doi:10.1073/pnas.1212070110.
  15. ↑ Saxena R, Fingland N, Patil D, Sharma AK, Crooke E (April 2013). "Crosstalk between DnaA protein, the initiator of Escherichia coli chromosomal replication, and acidic phospholipids present in bacterial membranes". International Journal of Molecular Sciences. 14 (4): 8517–37. PMC 3645759 Freely accessible. PMID 23595001. doi:10.3390/ijms14048517.
  16. ↑ McCarthy D, Minner C, Bernstein H, Bernstein C (October 1976). "DNA elongation rates and growing point distributions of wild-type phage T4 and a DNA-delay amber mutant". Journal of Molecular Biology. 106 (4): 963–81. PMID 789903. doi:10.1016/0022-2836(76)90346-6.
  17. ↑ Drake JW (1970) The Molecular Basis of Mutation. Holden-Day, San Francisco ISBN 0816224501 ISBN 978-0816224500
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