Eukaryotic DNA replication

Several factors are contributing to an increased air of excitement about the eukaryotic DNA replication problem: new insights into the nature of origins of replication, a better appreciation of the factors that control initiation, and studies of a DNA polymerase α-primase enzyme complex. In this review, recent research on the initiation, elongation and termination phases of DNA replication is critically examined and a coherent picture is formulated. In the not-far-distant future we expect to reproduce these processes in biochemically defined systems.     

Eukaryotic DNA replication.

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998     

Eukaryotic DNA replication reconstituted outside the cell

Our potential for dissecting the complex processes involved in eukaryotic DNA replication has been dramatically increased with the recent development of cell-free systems that recreate many of these processes in vitro. Initial results from these systems have drawn together work on the cell cycle, the enzymology of replication, and the structure of the nucleus.     

Eukaryotic error-prone DNA polymerases: The presumed roles in replication, repair, and mutagenesis

A number of error-prone DNA polymerases have been found in various eukaryotes, ranging from yeasts to mammals, including humans. According to partial homology of the primary structure, they are grouped into families B, X, and Y. These enzymes display a high infidelity on an intact DNA template, but they are accurate on a damaged template. Error-prone DNA polymerases are characterized by probabilities of base substitution or frameshift mutations ranging from 10^?3 to 7.5 · 10^?1 in an intact DNA, whereas the spontaneous mutagenesis rate per replicated nucleotide varies between 10^?10 and 10^?12. Low-fidelity polymerases are terminal deoxynucleotidyl transferase (TdT) and DNA polymerases β, ζ, κ, η, ι, λ, μ, and Rev1. The main characteristics of these enzymes are reviewed. None of them exhibits proofreading 3′ → 5′ exonuclease (PE) activity. The specialization of these polymerases consists in their capacity for synthesizing opposite DNA lesions (not eliminated by the numerous repair systems), which is explained by the flexibility of their active centers or a limited ability to express TdT activity. Classic DNA polymerases α, δ, ε, and γ cannot elongate primers with mismatched nucleotides at the 3′-end (which leads to replication block), whereas some specialized polymerases can catalyze this elongation. This is accompanied by overcoming the replication block, often at the expense of an increased mutagenesis rate. How can a cell exist under the conditions of this high infidelity of many DNA polymerase activities? Not all tissues of the body contain a complete set of low-fidelity DNA polymerases, although some of these enzymes are vitally important. In addition, cells “should not allow” error-prone DNA polymerases to work on undamaged DNA. After a lesion on the DNA template is bypassed, the cell should switch over from DNA synthesis catalyzed by specialized polymerases to the synthesis catalyzed by relatively high-fidelity DNA polymerases δ and ? (with an error frequency of 10^?5 to 10^?6) as soon as possible. This is done by forming complexes of polymerase δ or ? with proliferating cell nuclear antigen (PCNA) and replication factors RP-A and RF-C. These highly processive complexes show a greater affinity to correct primers than specialized DNA polymerases do. The fact that specialized DNA polymerases are distributive or weakly processive favors the switching. The fidelity of these polymerases is increased by the PE function of DNA polymerases δ and ε, as well as autonomous 3′ → 5′ exonucleases, which are widespread over the entire phylogenetic tree of eukaryotes. The exonuclease correction decelerates replication in the presence of lesions in the DNA template but increases its fidelity, which decreases the probability of mutagenesis and carcinogenesis.     

Eukaryotic DNA replication: from pre-replication complex to initiation complex

A common mechanism has emerged for the control of the initiation of eukaryotic DNA replication. The minichromosome maintenance protein complex (MCM) and Cdc45 have now been recognized as central components of the initiation machinery. In addition, two types of S phase promoting kinases conserved between yeast and humans play critical roles in the initiation reaction. At the onset of S phase, S phase kinases promote the association of Cdc45 with MCM at origins. Upon the formation of the MCM-Cdc45 complex at origins, the duplex DNA is unwound and various replication proteins, including DNA polymerases, are recruited onto unwound DNA. The increasing number of newly identified factors involved in the initiation reaction indicates that the control of initiation requires highly evolved machinery in eukaryotic cells.     

Eukaryotic error prone DNA polymerases: suggested roles in replication, repair and mutagenesis

A number of error-prone DNA polymerases is found among eukaryotes from yeasts up to mammalia including humans. According to the partial homology of a primary structure, they are united in families B, X, Y and display high infidelity on uninjured DNA-template, whereas they are rather accurate on DNA injuries. These DNA polymerases are characterized by the probability of base substitutions or frame shifts of 10(-3) to 7.5 x 10(-1) on DNA injuries, whereas the probability of spontaneous mutagenesis per replicated nucleotide accounts 10(-10) - 10(-12). Inaccurate DNA polymerases are terminal deoxynucleotidyl transferase (TdT), DNA polymerases beta, zeta, kappa, eta, iota, lamda, mu, and Rev1. Their principal properties are described in this review. All of the polymerases under study are deprived of the corrective 3'-->5' exonucleolytic activity. The specialization of these polymerases is contained in the capability to synthesize opposite DNA lesions (not eliminated by multiple repair systems) that is explained by the flexibility of their active sites or by the limited capability to exhibit the TdT activity. Classic DNA polymerases alpha, delta, epsilon, and gamma cannot elongate the primers with mismatched nucleotides on their 3'-ends (that leads to the replication block), whereas some of the specialized polymerases can do it. It is accompanied by the overcoming of a replication block, often with the expense of an elevated mutagenesis. How can a cell live under the conditions of such a huge infidelity of many DNA polymerases? Error-prone DNA polymerases are not found in all tissues though some of them are essential for an organism survival. Furthermore, cells must not allow for these polymerases to work effectively on uninjured DNA. After bypass of a lesion on DNA-template, it is necessary, as soon as possible, to switch catalysis of the DNA synthesis from the specialized polymerases on the relatively accurate DNA polymerases delta and epsilon (fidelity of 10(-5) - 10(-6)). It is made by the formation of the complexes of polymerases delta or epsilon with PCNA and replicative factors RP-A and RF-C. Such highly processive complexes manifest the bigger affinity to the correct primers than the specialized DNA polymerases do. The switching is stimulated by distributivity or weak processivity of the specialized DNA polymerases. The accuracy of these polymerases are augmented by the action of the corrective 3'-exonucleolytic function of DNA polymerases delta and epsilon as well as by the autonomous 3'-->5' exonucleases which are widespread among the representatives of the whole phylogenetic tree. Exonucleolytic correction slows down the replication in the presence of lesions in DNA-template but makes the replication more accurate that decreases the probability of mutagenesis and carcinogenesis.     

Eukaryotic DNA replication: a model for a fixed double replisome

To duplicate their genomes, eukaryotic cells have to overcome some formidable chemical and topological hurdles, considering the number of nucleotides that have to be polymerized faithfully and the sheer physical size of the DNA molecules that have to be disentangled and partitioned in an orderly way. This article tackles one particular aspect of the process: the organization of the apparatus that advances the replicative growing forks along the DNA molecule. Here, I suggest a solution to the difficulty of separating the daughter molecules in an orderly way and propose an alternative to the current models, which reconciles the use of a single polarity of synthesis by the DNA polymerases with the need for parallel polymerization of two strands of opposite polarity.     

Eukaryotic DNA metabolism : Are deoxyribonucleotides channeled to replication sites?

DNA precursor biosynthesis is closely coordinated with DNA replication itself. In prokaryotic systems, firm evidence supports the idea that this coordination is achieved through the action of multienzyme complexes that physically link the synthesis of deoxyribonucleotides with their utilization in DNA replication. Much evidence favors a similar channeling mechanism in eukaryotes. However, recent studies suggest strongly that in mammalian cells DNA precursors are synthesized in cytoplasm and are then transported into the nucleus. This article reviews the pertinent evidence, attempts to reconcile contradictory findings, and highlights areas that need further investigation.     

Eukaryotic genome instability in light of asymmetric DNA replication

The eukaryotic nuclear genome is replicated asymmetrically, with the leading strand replicated continuously and the lagging strand replicated as discontinuous Okazaki fragments that are subsequently joined. Both strands are replicated with high fidelity, but the processes used to achieve high fidelity are likely to differ. Here we review recent studies of similarities and differences in the fidelity with which the three major eukaryotic replicases, DNA polymerases α, δ, and ?, replicate the leading and lagging strands with high nucleotide selectivity and efficient proofreading. We then relate the asymmetric fidelity at the replication fork to the efficiency of DNA mismatch repair, ribonucleotide excision repair and topoisomerase 1 activity.