Single strand DNA binding and annealing activities in the yeast recombination factor Rad59

Saccharomyces cerevisiae RAD59 gene is required for homologous recombination processes and normal level of resistance to ionizing radiation. To study the biochemical functions of Rad59, it was overproduced in yeast and purified to near homogeneity. Rad59 binds DNA, showing much higher affinity for ssDNA than dsDNA. Rad59 also anneals complementary DNA strands, and order of addition experiments indicate that maximal annealing efficiency is achieved when both complementary DNA strands are present upon addition of Rad59. Thus, Rad59 resembles its homolog Rad52 in being able to bind ssDNA and anneal complementary DNA strands. However, unlike Rad52, DNA annealing by Rad59 is not accelerated by the ssDNA binding factor RPA. DNA binding and strand annealing are likely to be important for the biological functions of Rad59 in general recombination and in the single-strand annealing pathway of recombination.     

Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing

Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.     

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.     

Okazaki fragment maturation in yeast. I. Distribution of functions between FEN1 AND DNA2

In the presence of proliferating cell nuclear antigen, yeast DNA polymerase delta (Pol delta) replicated DNA at a rate of 40-60 nt/s. When downstream double-stranded DNA was encountered, Pol delta paused, but most replication complexes proceeded to carry out strand-displacement synthesis at a rate of 1.5 nt/s. In the presence of the flap endonuclease FEN1 (Rad27), the complex carried out nick translation (1.7 nt/s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primers. Downstream DNA primers, RNA primers, and small 5'-flaps were efficiently matured by Pol delta and FEN1, and Dna2 did not stimulate maturation. However, maturation of long 5'-flaps to which replication protein A can bind required both DNA2 and FEN1. The maturation kinetics were optimal with a slight molar excess over DNA of Pol delta, FEN1, and proliferating cell nuclear antigen. A large molar excess of DNA ligase substantially enhanced the rate of maturation and shortened the nick-translation patch (nucleotides excised past the RNA/DNA junction before ligation) to 4-6 nt from 8-12 nt with equimolar ligase. These results suggest that FEN1, but not DNA ligase, is a stable component of the maturation complex.     

Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G(2) phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation.     

The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes

Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5'-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes' activities.     

DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis

Brh2 is the Ustilago maydis ortholog of the BRCA2 tumor suppressor. It functions in repair of DNA by homologous recombination by controlling the action of Rad51. A critical aspect in the control appears to be the recruitment of Rad51 to single-stranded DNA regions exposed as lesions after damage or following a disturbance in DNA synthesis. In previous experimentation, Brh2 was shown to nucleate formation of the Rad51 nucleoprotein filament that becomes the active element in promoting homologous pairing and DNA strand exchange. Nucleation was found to be initiated at junctions of double-stranded and single-stranded DNA. Here we investigated the DNA binding specificity of Brh2 in more detail using oligonucleotide substrates. We observed that Brh2 prefers partially duplex structures with single-stranded branches, flaps, or D-loops. We found also that Brh2 has an inherent ability to promote DNA annealing and strand exchange reactions on free as well as RPA-coated substrates. Unlike Rad51, Brh2 was able to promote DNA strand exchange when preincubated with double-stranded DNA. These findings raise the notion that Brh2 may have roles in homologous recombination beyond the previously established Rad51 mediator activity.     

Structure of monoubiquitinated PCNA: Implications for DNA polymerase switching and Okazaki fragment maturation

Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer. Structurally, however, information on how ub-PCNA orchestrates the switching of these two classes of polymerases is lacking. We have solved the structure of ub-PCNA and demonstrate that the ubiquitin molecules in ub-PCNA are radially extended away from the PCNA without structural contact aside from the isopeptide bond linkage. This unique orientation provides an open platform for the recruitment of TLS polymerases through ubiquitin-interacting domains. However, the ubiquitin moieties, to the side of the equatorial PCNA plane, can place spatial constraints on the conformational flexibility of proteins bound to ub-PCNA. We show that ub-PCNA is impaired in its ability to support the coordinated actions of Fen1 and Pol δ in assays mimicking Okazaki fragment processing. This provides evidence for the novel concept that ub-PCNA may modulate additional DNA transactions other than TLS polymerase recruitment and switching.     

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation

Chromosomal DNA replication requires one daughter strand-the lagging strand-to be synthesised as a series of discontinuous, RNA-primed Okazaki fragments, which must subsequently be matured into a single covalent DNA strand. Here, we describe the reconstitution of Okazaki fragment maturation in vitro using proteins derived from the archaeon Sulfolobus solfataricus. Six proteins are necessary and sufficient for coupled DNA synthesis, RNA primer removal and DNA ligation. PolB1, Fen1 and Lig1 provide the required catalytic activities, with coordination of their activities dependent upon the DNA sliding clamp, proliferating cell nuclear antigen (PCNA). S. solfataricus PCNA is a heterotrimer, with each subunit having a distinct specificity for binding PolB1, Fen1 or Lig1. Our data demonstrate that the most efficient coupling of activities occurs when a single PCNA ring organises PolB1, Fen1 and Lig1 into a complex.     

The protein components and mechanism of eukaryotic Okazaki fragment maturation

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 delta 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.     

Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1

RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.     

Interaction and stimulation of human FEN-1 nuclease activities by heterogeneous nuclear ribonucleoprotein A1 in alpha-segment processing during Okazaki fragment maturation

High-fidelity DNA replication depends on both accurate incorporation of nucleotides in the newly synthesized strand and the maturation of Okazaki fragments. In eukaryotic cells, the latter is accomplished by a series of coordinated actions of a set of structure-specific nucleases, which, with the assistance of accessory proteins, recognize branched RNA/DNA configurations. In the current model of Okazaki fragment maturation, displacement of a 27-nucleotide or longer flap is envisioned to attract replication protein A (RPA), which inhibits flap endonuclease-1 (FEN-1) but stimulates Dna2 nuclease for cleavage. Dna2 cleavage generates a short flap of 5-7 nucleotides, which resists binding by RPA and further cleavage by Dna2. FEN-1 then removes the remaining flap to produce a suitable substrate for ligation. However, FEN-1 is not efficient in cleaving the short flap, and we therefore set out to identify cellular factors that might regulate FEN-1 activity. Through co-immunoprecipitation experiments, we have isolated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which forms a direct complex with FEN-1 and stimulates its enzymatic activities. The stimulation by hnRNP A1 is most dramatic using DNA substrates with short flaps. With longer flap substrates the hnRNP A1 effect is more modest and is suppressed by the addition of RPA. A model is provided to explain the possible in vivo role of this interaction and activity in Okazaki fragment maturation.     

DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein.

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.     

Effect of polyamine deficiency on proteins involved in Okazaki fragment maturation

Polyamine depletion causes S phase prolongation, and earlier studies indicate that the elongation step of DNA replication is affected. This led us to investigate the effects of polyamine depletion on enzymes crucial for Okazaki fragment maturation in the two breast cancer cell lines MCF-7 and L56Br-C1. In MCF-7 cells, treatment with N(1),N(11)-diethylnorspermine (DENSPM) causes S phase prolongation. In L56Br-C1 cells the prolongation is followed by massive apoptosis. In the present study we show that L56Br-C1 cells have substantially lower basal expressions of two Okazaki fragment maturation key proteins, DNA ligase I and FEN1, than MCF-7 cells. Thus, these two proteins might be promising markers for prediction of polyamine depletion sensitivity, something that can be useful for cancer treatment with polyamine analogues. DENSPM treatment affects the cellular distribution of FEN1 in L56Br-C1 cells, but not in MCF-7 cells, implying that FEN1 is affected by or involved in DENSPM-induced apoptosis.     

Homologous recombination via synthesis-dependent strand annealing in yeast requires the Irc20 and Srs2 DNA helicases

Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template, while maintaining the original configuration of the flanking regions. In somatic cells at the 4n stage, Holliday-junction-mediated homologous recombination and nonhomologous end joining (NHEJ) cause crossovers (CO) between homologous chromosomes and deletions, respectively, resulting in loss of heterozygosity (LOH) upon cell division. However, the SDSA pathway prevents DSB-induced LOH. We developed a novel yeast DSB-repair assay with two discontinuous templates, set on different chromosomes, to determine the genetic requirements for somatic SDSA and precise end joining. At first we used our in vivo assay to verify that the Srs2 helicase promotes SDSA and prevents imprecise end joining. Genetic analyses indicated that a new DNA/RNA helicase gene, IRC20, is in the SDSA pathway involving SRS2. An irc20 knockout inhibited both SDSA and CO and suppressed the srs2 knockout-induced crossover enhancement, the mre11 knockout-induced inhibition of SDSA, CO, and NHEJ, and the mre11-induced hypersensitivities to DNA scissions. We propose that Irc20 and Mre11 functionally interact in the early steps of DSB repair and that Srs2 acts on the D-loops to lead to SDSA and to prevent crossoverv.     

Okazaki fragment maturation in yeast. II. Cooperation between the polymerase and 3'-5'-exonuclease activities of Pol delta in the creation of a ligatable nick

To address the different functions of Pol delta and FEN1 (Rad27) in Okazaki fragment maturation, exonuclease-deficient polymerase Pol delta-01 and Pol delta-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), respectively) were purified and characterized in this process. In the presence of the replication clamp PCNA, both wild-type and exo(-) Pol delta carried out strand displacement synthesis with similar rates; however, initiation of strand displacement synthesis was much more efficient with Pol delta-exo(-). When Pol delta-exo(-) encountered a downstream primer, it paused with 3-5 nucleotides of the primer displaced, whereas the wild type carried out precise gap filling. Consequently, in the absence of FEN1, Pol delta exonuclease activity was essential for closure of simple gaps by DNA ligase. Compared with wild type, Okazaki fragment maturation with Pol delta-exo(-) proceeded with an increased duration of nick translation prior to ligation. Maturation was efficient in the absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In agreement with these results, the proposed generation of double strand breaks in pol3-exo(-) rad27 mutants was suppressed by the overexpression of DNA2. Further genetic studies showed that pol3-exo(-) rad27 double mutants were sensitive to alkylation damage consistent with an in vivo defect in gap filling by exonuclease-deficient Pol delta.