Eukaryotic DNA Replication Fork

Abstract

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Keywords: CMG helicase; DNA polymerase; DNA primase; Okazaki fragment; replisome coordination.

Figure 1

Assembly of the eukaryotic replisome. The origin-bound ORC–Cdc6 complex initially recruits one Cdt1–Mcm2-7 complex, followed by a second complex, to form a double Mcm2-7 hexamer. Further assembly requires Dpb11, Sld2, Sld3, and Sld7, which are not thought to be associated with the mature replisome, and Cdc45, GINS, and Pol ε, which are associated with it, as well as DDK and CDK kinase activity to complete assembly and prime the complex for helicase activation that is accomplished by Mcm10 and RPA. Abbreviations: CDK, cyclin-dependent kinase; DDK, Cdc7/Dbf4 kinase; GINS, Sld5, Psf1, Psf2, and Psf3 complex; Mcm2-7, helicase complex; ORC, origin recognition complex; Pol, DNA polymerase; RPA, replication protein A.

Figure 2

Strand-specific mapping techniques. (a) Mapping of strand-specific protein binding. Replicating cells are pulse labeled with bromodeoxyuridine (BrdU), followed by chromatin-immunoprecipitation (ChIP) of a lagging strand–associated protein. Protein-associated nascent single-stranded DNA (ssDNA) is enriched by immunoprecipitation (IP) with antibodies against BrdU, and the isolated ssDNA is subjected to strand-specific sequencing. The sequence reads are mapped to both the Watson (W) and Crick (C) strands and plotted as a ratio of W/C reads. The opposite result is expected when the experiment is carried out with a protein associated with the nascent leading strand. (b) Mapping of ribonucleotide monophosphate (rNMP) incorporation by the rNMP-prone lagging strand polymerase variant. The frequent rNMP incorporation by DNA polymerase δ (L612M) was detected in an RNH201 strain that eliminates ribonucleotide excision repair. After cleavage of ribonucleotides in the isolated DNA with alkali or ribonuclease H2 (RNase H2), various technologies have been used to target these ends (either the ribose-2′- or 3′-phosphate end or the 5′-phosphate end) for strand-specific sequencing. The sequence reads are mapped to both the Watson (W) and Crick (C) strands and plotted as a ratio of W/C reads. The opposite result is expected when the experiment is carried out with the rNMP-prone leading strand polymerase variant.

Figure 3

Eukaryotic DNA replicases. DNA polymerase α (Pol α, red ) and Pol ε ( green) contain four subunits, and Saccharomyces cerevisiae Pol δ (blue) contains three subunits, whereas human Pol δand Schizosaccharomyces pombe Pol δhave an additional small fourth subunit (not shown). Demonstrated [4Fe–4S] iron–sulfur clusters are indicated with large orange balls, and bound zinc atoms with small gray balls. Catalytic properties and protein–protein interactions are listed. Note that Pol δhas a high fidelity for base–base mismatches but lower fidelity for single-nucleotide deletions in repetitive sequences. Abbreviations: GINS, Sld5, Psf1, Psf2, and Psf3 complex; n.d., not determined; PCNA, proliferating cell nuclear antigen.

Figure 4

Replisome structure and interactions. Two models for the pathway taken by the leading strand prior to entry into the DNA polymerase ε (Pol ε) catalytic site. Either (a) ~40-nt or (b) ~20-nt lengths of single-stranded DNA are occluded. The proposal has been made that these two forms can also interconvert. The lagging strand is shown looped such that both Pol α and Pol ε move in the same direction while held in a complex by Ctf4. Abbreviations: GINS, Sld5, Psf1, Psf2, and Psf3 complex; Mcm2-7, helicase complex; PCNA, proliferating cell nuclear antigen; RPA, replication protein A.

Figure 5

Okazaki fragment maturation. Primers on the lagging strand are elongated by Pol δ (DNA polymerase δ) until the downstream Okazaki fragment is reached. Subsequent strand displacement synthesis by Pol δ is counteracted by its 3′-exonuclease activity (idling). In the presence of FEN1, the nascent flap is cut and strand displacement synthesis restarts. This iterative process (nick translation) predominantly releases mononucleotides. Occasional excess strand displacement synthesis yields very long 5′-flaps that are processed to short flaps by the nuclease activity of Dna2. After degradation of all primer RNA, ligation of the DNA–DNA nick is performed by DNA ligase 1. Abbreviations: FEN1, 5′-flap endonuclease 1; PCNA, proliferating cell nuclear antigen; RPA, replication protein A.