DNA polymerases that propagate the eukaryotic DNA replication fork

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase ot-primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase ca and recruits DNA polymerase S and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase 8, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute.     

The Protein Components and Mechanism of Eukaryotic Okazaki Fragment Maturation

ABSTRACT

An initiator RNA (iRNA) is required to prime cellular DNA synthesis. The structure of double-stranded DNA allows the synthesis of one strand to be continuous but the other must be generated discontinuously. Frequent priming of the discontinuous strand results in the formation of many small segments, designated Okazaki fragments. These short pieces need to be processed and joined to form an intact DNA strand. Our knowledge of the mechanism of iRNA removal is still evolving. Early reconstituted systems suggesting that the removal of iRNA requires sequential action of RNase H and flap endonuclease 1 (FEN1) led to the RNase H/FEN1 model. However, genetic analyses implied that Dna2p, an essential helicase/nuclease, is required. Subsequent biochemical studies suggested sequential action of RPA, Dna2p, and FEN1 for iRNA removal, leading to the second model, the Dna2p/RPA/FEN1 model. Studies of strand-displacement synthesis by polymerase δ indicated that in a reconstituted system, FEN1 could act as soon as short flaps are created, giving rise to a third model, the FEN1-only model. Each of the three pathways is supported by different genetic and biochemical results. Properties of the major protein components in this process will be discussed, and the validity of each model as a true representation of Okazaki fragment processing will be critically evaluated in this review.     

An alternative pathway for Okazaki fragment processing: resolution of fold-back flaps by Pif1 helicase

Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo.     

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer.     

An updated perspective on the polymerase division of labor during eukaryotic DNA replication

Abstract

In eukaryotes three DNA polymerases (Pols), α, δ, and ε, are tasked with bulk DNA synthesis of nascent strands during genome duplication. Most evidence supports a model where Pol α initiates DNA synthesis before Pol ε and Pol δ replicate the leading and lagging strands, respectively. However, a number of recent reports, enabled by advances in biochemical and genetic techniques, have highlighted emerging roles for Pol δ in all stages of leading-strand synthesis; initiation, elongation, and termination, as well as fork restart. By focusing on these studies, this review provides an updated perspective on the division of labor between the replicative polymerases during DNA replication.     

How the Cell Deals with DNA Nicks

During lagging strand DNA replication, the Okazaki fragment maturation machinery is requiredto degrade the initiator RNA with high speed and efficiency, and to generate with great accuracya proper DNA nick for closure by DNA ligase. Several operational parameters are important ingenerating and maintaining a ligatable nick. These are the strand opening capacity of the laggingstrand DNA polymerase ? (Pol ?), and its ability to limit strand opening to that of a fewnucleotides. In the presence of the flap endonuclease FEN1, Pol ? rapidly hands off the strandopenedproduct for cutting by FEN1, while in its absence, the ability of DNA polymerase ? toswitch to its 3’-5’-exonuclease domain in order to degrade back to the nick position is importantin maintaining a ligatable nick. This regulatory system has a built-in redundancy so thatdysfunction of one of these activities can be tolerated in the cell. However, further dysfunctionleads to uncontrolled strand displacement synthesis with deleterious consequences, as is revealedby genetic studies of exonuclease-defective mutants of S. cerevisiae Pol ?. These sameparameters are also important for other DNA metabolic processes, such as base excision repair,that depend on Pol ? for synthesis.     

The hyperthermophilic euryarchaeota Pyrococcus abyssi likely requires the two DNA polymerases D and B for DNA replication

DNA polymerases carry out DNA synthesis during DNA replication, DNA recombination and DNA repair. During the past five years, the number of DNA polymerases in both eukarya and bacteria has increased to at least 19 and multiple biological roles have been assigned to many DNA polymerases. Archaea, the third domain of life, on the other hand, have only a subset of the eukaryotic-like DNA polymerases. The diversity among the archaeal DNA polymerases poses the intriguing question of their functional tasks. Here, we focus on the two identified DNA polymerases, the family B DNA polymerase B (PabpolB) and the family D DNA polymerase D (PabpolD) from the hyperthermophilic euryarchaeota Pyrococcus abyssi. Our data can be summarized as follows: (i) both Pabpols are DNA polymerizing enzymes exclusively; (ii) their DNA binding properties as tested in gel shift competition assays indicated that PabpolD has a preference for a primed template; (iii) PabPolD is a primer-directed DNA polymerase independently of the primer composition whereas PabpolB behaves as an exclusively DNA primer-directed DNA polymerase; (iv) PabPCNA is required for PabpolD to perform efficient DNA synthesis but not PabpolB; (v) PabpolD, but not PabpolB, contains strand displacement activity; (vii) in the presence of PabPCNA, however, both Pabpols D and B show strand displacement activity; and (viii) we show that the direct interaction between PabpolD and PabPCNA is DNA-dependent. Our data imply that PabPolD might play an important role in DNA replication likely together with PabpolB, suggesting that archaea require two DNA polymerases at the replication fork.     

Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis

Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase delta (Pol delta) with 5.-flap cutting by FEN1(RAD27) endonuclease. Excessive strand displacement is normally prevented by the 3.-exonuclease activity of Pol delta. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol delta) or PIF1 helicase genes can suppress lethality or growth defects of rad27Delta pol3-D520V mutants (defective for FEN1(RAD27) and the 3.-exonuclease of Pol delta) that produce long flaps and of dna2Delta mutants that are defective in cutting long flaps. On the contrary, pol32Delta or pif1Delta caused lethality of rad27Delta exo1Delta double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1(RAD27) and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol delta is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1(RAD27) during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol delta to FEN1(RAD27). Finally, RNA-DNA hybrids are more readily displaced by Pol delta than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.     

Polymerase dynamics at the eukaryotic DNA replication fork

This review discusses recent insights in the roles of DNA polymerases (Pol) delta and epsilon in eukaryotic DNA replication. A growing body of evidence specifies Pol epsilon as the leading strand DNA polymerase and Pol delta as the lagging strand polymerase during undisturbed DNA replication. New evidence supporting this model comes from the use of polymerase mutants that show an asymmetric mutator phenotype for certain mispairs, allowing an unambiguous strand assignment for these enzymes. On the lagging strand, Pol delta corrects errors made by Pol alpha during Okazaki fragment initiation. During Okazaki fragment maturation, the extent of strand displacement synthesis by Pol delta determines whether maturation proceeds by the short or long flap processing pathway. In the more common short flap pathway, Pol delta coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase.     

Replication dynamics in fission and budding yeasts through DNA polymerase tracking

The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof‐read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions.     

Comparison of functional properties of mammalian DNA polymerase lambda and DNA polymerase beta in reactions of DNA synthesis related to DNA repair

DNA polymerase lambda (Pol lambda) is a novel enzyme of the family X of DNA polymerases. Pol lambda has some properties in common with DNA polymerase beta (Pol beta). The substrate properties of Pol lambda were compared to Pol beta using DNAs mimicking short-patch (SP) and long-patch (LP) base excision repair (BER) intermediates as well as recessed template primers. In the present work, the influence of several BER proteins such as flap-endonuclease-1 (FEN1), PCNA, and apurinic/apyrimidinic endonuclease-1 (APE1) on the activity of Pol lambda was investigated. Pol lambda is unable to catalyze strand displacement synthesis using nicked DNA, although this enzyme efficiently incorporates a dNMP into a one-nucleotide gap. FEN1 and PCNA stimulate the strand displacement activity of Pol lambda. FEN1 processes nicked DNA, thus removing a barrier to Pol lambda DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol lambda. Photocrosslinking and functional assay show that Pol lambda is less efficient than Pol beta in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol lambda though it stimulates strand displacement synthesis catalyzed with Pol beta. It is suggested that Pol lambda plays a role in the SP BER rather than contributes to the LP BER pathway.     

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

DNA polymerase epsilon interacts with the CMG (Cdc45-MCM-GINS) complex by Dpb2p, the non-catalytic subunit of DNA polymerase epsilon. It is postulated that CMG is responsible for targeting of Pol ɛ to the leading strand. We isolated a mutator dpb2-100 allele which encodes the mutant form of Dpb2p. We showed previously that Dpb2-100p has impaired interactions with Pol2p, the catalytic subunit of Pol ɛ. Here, we present that Dpb2-100p has strongly impaired interaction with the Psf1 and Psf3 subunits of the GINS complex. Our in vitro results suggest that while dpb2-100 does not alter Pol ɛ’s biochemical properties including catalytic efficiency, processivity or proofreading activity – it moderately decreases the fidelity of DNA synthesis. As the in vitro results did not explain the strong in vivo mutator effect of the dpb2-100 allele we analyzed the mutation spectrum in vivo. The analysis of the mutation rates in the dpb2-100 mutant indicated an increased participation of the error-prone DNA polymerase zeta in replication. However, even in the absence of Pol ζ activity the presence of the dpb2-100 allele was mutagenic, indicating that a significant part of mutagenesis is Pol ζ-independent. A strong synergistic mutator effect observed for transversions in the triple mutant dpb2-100 pol2-4 rev3Δ as compared to pol2-4 rev3Δ and dpb2-100 rev3Δ suggests that in the presence of the dpb2-100 allele the number of replication errors is enhanced. We hypothesize that in the dpb2-100 strain, where the interaction between Pol ɛ and GINS is weakened, the access of Pol δ to the leading strand may be increased. The increased participation of Pol δ on the leading strand in the dpb2-100 mutant may explain the synergistic mutator effect observed in the dpb2-100 pol3-5DV double mutant.     

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.     

In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication

The mechanisms of mitochondrial DNA replication have been hotly debated for a decade. The strand‐displacement model states that lagging‐strand DNA synthesis is initiated from the origin of light‐strand DNA replication (OriL), whereas the strand‐coupled model implies that OriL is dispensable. Mammalian mitochondria cannot be transfected and the requirements of OriL in vivo have therefore not been addressed. We here use in vivo saturation mutagenesis to demonstrate that OriL is essential for mtDNA maintenance in the mouse. Biochemical and bioinformatic analyses show that OriL is functionally conserved in vertebrates. Our findings strongly support the strand‐displacement model for mtDNA replication.     

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5′-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.     

Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max

A DNA helicase activity was detected in extracts of purified chloroplasts from the SB-1 cell line of Glycine max and partially purified by column chromatography on DEAE cellulose, phosphocellulose, and single-stranded DNA cellulose. The chloroplast helicase has a DNA-dependent ATPase activity, and its strand displacement activity is strictly dependent upon the presence of a nucleoside triphosphate and Mg²⁺ or Mn²⁺. Strand displacement activity does not require a free unannealed single-strand or replication fork-like structure.     

Exonuclease 1 preferentially repairs mismatches generated by DNA polymerase α

The Saccharomyces cerevisiae EXO1 gene encodes a 5′ exonuclease that participates in mismatch repair (MMR) of DNA replication errors. Deleting EXO1 was previously shown to increase mutation rates to a greater extent when combined with a mutator variant (pol3-L612M) of the lagging strand replicase, DNA polymerase δ (Pol δ), than when combined with a mutator variant (pol2-M644G) of the leading strand replicase, DNA polymerase ? (Pol ?). Here we confirm that result, and extend the approach to examine the effect of deleting EXO1 in a mutator variant (pol1-L868M) of Pol α, the proofreading-deficient and least accurate of the three nuclear replicases that is responsible for initiating Okazaki fragment synthesis. We find that deleting EXO1 increases the mutation rate in the Pol α mutator strain to a significantly greater extent than in the Pol δ or Pol ? mutator strains, thereby preferentially reducing the efficiency of MMR of replication errors generated by Pol α. Because these mismatches are closer to the 5′ ends of Okazaki fragments than are mismatches made by Pol δ or Pol ?, the results not only support the previous suggestion that Exo1 preferentially excises lagging strand replication errors during mismatch repair, they further imply that the 5′ ends serve as entry points for 5′ excision of replication errors made by Pol α, and possibly as strand discrimination signals for MMR. Nonetheless, mutation rates in the Pol α mutator strain are 5- to 25-fold lower in an exo1Δ strain as compared to an msh2Δ strain completely lacking MMR, indicating that in the absence of Exo1, most replication errors made by Pol α can still be removed in an Msh2-dependent manner by other nucleases and/or by strand displacement.