DNA Replication: Yeast

Abstract

DNA replication is a complex biological process essential to maintaining the fidelity of genetic information passed to subsequent generations, while allowing for the mutations necessary for natural selection. The cellular machinery required for DNA replication must be precisely controlled throughout the cycle. A variety of DNA polymerases have proofreading and repairing capabilities to avoid catastrophic errors, thus maintaining genomic stability. Much of the mechanism involved in this process is evolutionarily conserved. Because yeast is a relatively simple and inexpensive organism to work with, much of our understanding of DNA replication in eukaryotes originates from experiments with yeast. The most thoroughly characterized model yeast systems are Saccharomyces cerevisiae (budding yeast) and Saccharomyces pombe (fission yeast).

Key Concepts

  • Yeast is a good model organism for simplifying and characterising cellular processes, such as DNA replication.
  • The two most commonly used yeast models are S. cerevisiae and S. pombe.
  • DNA replication is a complex biological process essential to genomic replication.
  • DNA replication is a process that involves timely organisation of proteins and is regulated during the cell cycle by cyclin‚Äźdependent protein kinases.
  • Termination of DNA replication in yeast is generally not sequence specific, except in special circumstances in the cellular process.

Keywords: Saccharomyces cerevisiae; Saccharomyces pombe; cell cycle; initiation; elongation; termination; replication complex; autonomous replicating sequence (ARS)

Figure 1. Initiation of DNA replication in S. cerevisiae. The DNA replication origins in S. cerevisiae are autonomously replicating sequences (ARS) consisting of element A (a conserved 11 bp AT‐rich sequence or ACS), B1, and B2 on the DNA. Element A and B1 is required for ORC binding. B2 is proposed to have a dsDNA unwinding element that is needed for MCM binding. Some origins have element B3, where the transcription factor Abf1 binds. At the G1 phase, CDK levels are low, and the double hexamer of MCM is inactive. Binding of inactive MCM (Mcm2‐7) requires ORC, Cdc6 and Cdt1. At high CDK levels, the double hexamer MCM is activated as the core component of the CMG complex. The CMG complex consists of: Cdc45, MCM and GINS (Sld5, Psf1, Psf2 and Psf3).
Figure 2. DNA duplex unwinding and replication fork formation in S. pombe. Origin recognition complex (ORC) associated with chromatin marks the ARS elements. Upon the initiation of replication, the MCM recruits Cdc18 (homologue of S. cerevisiae Cdc 6) and Cdt1 to the origin, thereby forming the prereplicative complex (pre‐RC). At the G1 – S transition, Sna41 (homologue of S. cerevisiae Cdc45) associates with the pre‐RC as a result of the action of several protein kinases (not shown) forming the preinitiation complex (pre‐IC). In the S phase, the two activated CMG helicase structures will move in opposite directions down the dsDNA strand, unwinding dsDNA via ATP hydrolysis, and creating two DNA replication forks.
Figure 3. Simplified model of leading‐ and lagging‐strand synthesis during elongation. DNA synthesis moves away from the DNA replication origin in two opposite DNA replication forks. As the CMG complex (Cdc45, MCM2‐7, GINS) opens double‐helical DNA, topoisomerases relieve torsional strain in DNA. Yellow bars represent RNA–DNA primers synthesised by Pol α. DNA synthesis by Pol ϵ at the leading strand is increased in the presence of PCNA. Pol δ is the major polymerase that synthesises DNA at the lagging strand and is very processive in the presence of PCNA. Many molecules of RPA bind to exposed ssDNA.
Figure 4. Comparison of the programmed termination of rDNA in S. cerevisiae and S. pombe. One complete origin to origin unit is shown for each type of yeast. (a) In S. cerevisiae, Fob1 binds to identical Ter sequences on the rDNA strand. (b) In S. pombe, Sap1 binds to Ter1, while Reb1 binds to Ter2 and Ter3 on the rDNA strand. An additional factor, Rtf1, has been shown bind to Ter site RTS1. The direction of replication is shown with black arrows below each strand. The T‐bar in the opposite direction indicates replication fork stalling.
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Further Reading

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Tan C and Tomkins J (2015) Information processing differences between bacteria and eukarya – Implications for the myth of eukaryogenesis. Answers Research Journal 8: 143–162.

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Smith, Shanna J, Li, Caroline M, Raoof, Mustafa, Lingeman, Robert G, Hickey, Robert J, and Malkas, Linda H(Sep 2018) DNA Replication: Yeast. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0027975]