DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces cerevisiae

Abstract

This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity.

Keywords: DNA helicases; DNA replication; Okazaki fragment processing; flap endonucleases; lagging strand DNA synthesis; strand displacement DNA synthesis.

Figure 1

Schematic structure of the replisome and the DNA replication fork of S. cerevisiae. DNA polymerase ε synthesizes the leading strand continuously while DNA polymerase α-Primase and DNA polymerase δ carry out lagging strand synthesis in a discontinuous way creating Okazaki fragments (OFs). DNA polymerase α-Primase makes the RNA primer and synthesizes the DNA initiator fragment. After this event, Pol α falls off from the 3’ end of the growing OF at the replication fork and PCNA (Proliferating Cell Nuclear Antigen) is loaded by the RFC (Replication Factor C) complex leading to a DNA polymerase switch between Pol α and Pol δ on the lagging strand. The growing 3’ end of the previous OF, which is being synthesized by Pol δ will encounter the 5’ end of the last OF, which contains the RNA-DNA initiators segments previously synthesized by Pol α. This encounter will lead to the processing and maturation of the OF (see Figure 2).

Figure 2

Possible mechanisms of Okazaki fragments (OF) maturation in S. cerevisiae. (A) Direct removal of the RNA primer by RNase H2 combined with limited Pol δ-mediated DNA synthesis leads to the formation of a ligatable nick, which is a substrate for DNA ligase I. (B) Limited strand displacement of the 5’ end of the last OF by Pol δ (due to DNA polymerase idling) displaces a single nucleotide that is cut by Fen1 or Exo1. Iterative cycles of Pol δ-Fen1 or Pol δ-Exo1-dependent nick translation will remove the RNA primer and create a ligatable nick for DNA ligase I. (C) More cycles of nick translation will remove also the initiator DNA previously synthesized by Pol α. (D) Extended Pif1-PCNA-Pol δ-dependent strand displacement DNA synthesis proceeds beyond the initiator DNA. The displaced 5’ end cannot be cut by Fen1 and Exo1 and requires processing by Dna2. Once Dna2 has reduced the length of the displaced tail, short flaps become substrates for Fen1 or Exo1, which will create a ligatable nick for DNA ligase I (note sequential actions of Dna2 and Fen1). (E) In the absence of Fen1, Pif1-PCNA-Pol δ-dependent long flaps are shortened by Dna2 to a length that only sporadically allows the binding of RPA and DDR activation (also because Exo1 can back up Fen1 on short flaps, so that rad27 cells can support viability; see the text). (F) Small flaps produced in the absence of Fen1 may also be repaired through homologous recombination with the sister chromatid. This mechanism could explain why RAD27 mutations are lethal in combination with homologous recombination mutations. (G) In the absence of Dna2, Pif1-PCNA-Pol δ -dependent long flaps are not processed, and ssDNA bound by RPA remains exposed, Fen1-mediated cutting is inhibited and DDR hyper-activation is triggered. Exo1 or RNase H2 should not be able to cut long DNA flaps in dna2 cells and does not prevent DDR hyper-activation in dna2 defective cells. DDR activation in Dna2 depleted cells leads to cell cycle arrest and cell death, strongly suggesting that the products of OF maturation in the absence of Dna2 are not compatible with viability.

Figure 3

DNA synthesis through strand displacement in bacterial and bacteriophage DNA polymerases. (A) E. coli DNA polymerase I (PolI) can carry out DNA synthesis through strand displacement on a nicked duplex linear DNA molecule. When PolI starts to extend the 3’ end of the nick there is a first phase of nick translation where the 5’ end of the nick is degraded by its 5’–3’ exonuclease activity. In this phase, there is no net DNA synthesis. A second phase of DNA synthesis through strand displacement follows in which the 5’–3’ exonuclease activity is inhibited and the 5’ end of the nick starts to be displaced in a ssDNA tail [111,112]. (B) The T4 DNA polymerase (T4Pol, gp43 protein) together with the T4 single strand binding protein (ssb, gp32 protein) and other structural factors (gp62, gp45 and gp44) can carry out extended DNA synthesis through strand displacement (in the absence of the T4 DNA helicase, gp41 protein) either on linear duplex or circular duplex nicked DNA templates. The activity of the T4 Pol on these substrates produces long displaced ssDNA tails [114].

Figure 4

(A) Backward directed Pif1-PCNA-Dna2-Pol δ mediated strand displacement DNA synthesis and flap cutting may be utilized to remove DNA replication obstacles such as non-nucleosomal DNA-protein complexes and R-loops created by the transcription units to counteract DNA replication fork pausing (see the text). (B) Pif1-Pol δ mediated D-loop bubble migration and extended strand displacement DNA synthesis coupled to Dna2-dependent cutting of the resulting DNA flaps during conservative BIR (break-induced replication) contribute to create a fully replicated duplex. (C) Backward directed Pif1-PCNA-Dna2-Pol δ-mediated strand displacement DNA synthesis and flap cutting may be utilized to remove flaps carrying DNA hairpins during lagging strand DNA replication of repetitive sequences.

Figure 5

(A) graphical map of the direct physical interactions of factors involved in OF processing. Different mechanisms of OF processing dictated by different structural architectures of the complex Pif1-PCNA-Dna2-FEN1-Pol δ macromolecular assembly (B–E).