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.     

Phylogenetic analysis of archaeal PCNA homologues

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages. Received: July 10, 2000 / Accepted: September 26, 2000     

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA^S228I mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3^p66 and PolD4^p12. In contrast p21 largely retains the ability to bind PCNA^S228I. This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA^S228I binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA^S228I for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.     

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 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.     

DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork

Abstract

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 α -primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase α and recruits DNA polymerase δ 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 δ, 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 ε normally replicates this strand, but under conditions of dysfunction, DNA polymerase δ may substitute.     

古菌RecJ蛋白的研究进展

RecJ蛋白属于aspartate-histidine-histidine (DHH)磷酸酯酶超家族,存在于细菌、真核生物和古菌中。细菌RecJ蛋白是一种5′→3′ssDNA外切酶,参与错配修复、同源重组、碱基切除修复等生物学过程。真核生物cell division cycle 45 (Cdc45)蛋白是细菌RecJ核酸酶的同源物,但不具有核酸酶活性。Cdc45蛋白能够与minichromosomemaintenance(MCM)和Go-Ichi-Ni-San(GINS)形成Cdc45-MCM-GINS (CMG)复合物,是真核生物DNA复制的重要组分。在古菌中,几乎所有基因组已测序的古菌均编码一种或多种RecJ蛋白同源物。与细菌RecJ核酸酶不同,古菌RecJ蛋白具有多样化的核酸酶活性,并且能够与MCM和GINS形成类似于真核生物CMG的复合物。因此,古菌RecJ蛋白是参与古菌DNA复制、修复和重组的重要成分。基于目前古菌RecJ蛋白的研究报道,本文综述了古菌RecJ蛋白的活性、结构与功能方面的研究进展,聚焦于不同古菌RecJ蛋白以及它们与细菌RecJ核酸酶和真核生物RecJ同源物的...     

RNA:DNA hybrids from Okazaki fragments contribute to establish the Ku-mediated barrier to replication-fork degradation

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DNA连接酶同工酶的研究进展

DNA连接酶是生物体内重要的酶,其所催化的反应在DNA的复制和修复过程中起重要作用. DNA连接酶分为两大类：一类是利用ATP的能量催化两个核苷酸链之间形成磷酸二酯键的依赖ATP的DNA连接酶,另一类是利用NAD⁺的能量催化两个核苷酸链之间形成磷酸二酯键的依赖NAD^＋的DNA连接酶.研究发现,细菌的DNA连接酶都是依赖NAD^＋的, 且有非常相似的序列和相近的分子质量,其酶分子分为两个功能区：N端区与NAD^＋结合形成酶-腺苷酸中间物;C端区催化两条DNA链的连接.所有真核生物的DNA连接酶都是利用ATP提供能量,且一种真核生物含有多种DNA连接酶,不同的DNA连接酶催化不同的DNA修复和复制过程：DNA连接酶Ⅰ的作用是将岗畸片段连接起来形成完整的DNA链以及进行碱基切除修复(BER);DNA连接酶Ⅲ主要是在DNA修复中起作用,即催化单核苷酸碱基切除修复.DNA连接酶Ⅱ可能是DNA连接酶Ⅲ的一个片段.     

Structure of monoubiquitinated PCNA

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.     

SUMO modification of PCNA is controlled by DNA

Post-translational modification by the ubiquitin-like protein SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, many SUMO-specific ligases do not exhibit much target specificity, and--compared with the diversity of sumoylation substrates--their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We report here an unexpected mechanism by which sumoylation of the replication clamp protein, PCNA, from budding yeast is effectively coupled to S phase. We find that loading of PCNA onto DNA is a prerequisite for sumoylation in vivo and greatly stimulates modification in vitro. To our surprise, however, DNA binding by the ligase Siz1, responsible for PCNA sumoylation, is not strictly required. Instead, the stimulatory effect of DNA on conjugation is mainly attributable to DNA binding of PCNA itself. These findings imply a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated.     

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.     

Cooperative regulation for Okazaki fragment processing by RNase HII and FEN-1 purified from a hyperthermophilic archaeon,Pyrococcus furiosus

A reconstitution system that recapitulates the processing of Okazaki-primer RNA was established by the heat-stable recombinant enzymes RNase HII and FEN-1 (termed Pf-RNase HII and Pf-FEN-1, respectively) prepared from a hyperthermophilic archaeon, Pyrococcus furiosus. A 35-mer RNA-DNA/DNA hybrid substrate mimicking an Okazaki fragment was used to investigate the properties of the processing reaction in vitro at 50 degrees C. Pf-RNase HII endonucleolytically cleaves the RNA primer region, but does not cut the junction between RNA and DNA. Removal of the RNA of the RNA-DNA junction was brought about by Pf-FEN-1 after Pf-RNase HII digestion. In the presence of 0.25-5mM MnCl(2), Pf-FEN-1 alone weakly cleaved the junction. The addition of Pf-RNase HII to the reaction mixture increased removal efficiency and optimal Pf-FEN-1 activity was achieved at an equal amount of the two enzymes. These results indicate that there are at least two steps in the degradation of primer RNA requiring a step-specific enzyme. It is likely that Pf-RNase HII and Pf-FEN-1 cooperatively process Okazaki fragment during lagging-strand DNA replication.     

The trans-autostimulatory activity of Rad27 suppresses dna2 defects in Okazaki fragment processing

Dna2 and Rad27 (yeast Fen1) are the two endonucleases critical for Okazaki fragment processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. In this study, we addressed the functional consequences of these interactions by examining whether purified Rad27 of Saccharomyces cerevisiae affects the enzymatic activity of Dna2 and vice versa. For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna2, but failed to do so with Dna2Δ405N that lacks the N-terminal 405 amino acids. This was an unexpected finding because dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-autostimulatory enzyme, providing an explanation why overexpression of Rad27, regardless of its catalytic activity, suppressed dna2 mutants as long as an endogenous wild type Rad27 is available. We found that the C-terminal 16-amino acid fragment of Rad27, a highly polybasic region due to the presence of multiple positively charged lysine and arginine residues, was sufficient and necessary for the stimulation of both Rad27 and Dna2. Our findings provide further insight into how Dna2 and Rad27 jointly affect the processing of Okazaki fragments in eukaryotes.     

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.     

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.     

Stabilization of an archaeal DNA-sliding clamp protein, PCNA, by proteasome-activating nucleotidase gene knockout in Haloferax volcanii

Many details of structure, function and substrate specificity of eukaryotic proteasomal systems have been elucidated. This information far-exceeds that available for the archaeal and bacterial counterparts. While structural and functional studies have provided some insight into the workings of prokaryotic proteasomes, the question of substrate targeting and global cellular influence remain largely unaddressed. In this communication, we report an over 720-fold increase in the half-life of the DNA-sliding clamp protein proliferating cell nuclear antigen after knockout of the panA gene, encoding a proteasome-activating nucleotidase A, on the chromosome of the halophilic archaeon Haloferax volcanii . This discovery marks the first identification of a protein stabilized by an archaeal proteasome mutation and provides a starting point for investigations into substrate recognition mechanisms. The findings also begin to address the functional role of proteasomal systems within the scope of the archaeal cell.     

The acetylatable lysines of human Fen1 are important for endo- and exonuclease activities

Human Fen1 can be acetylated in vivo and in vitro resulting in reduced endonuclease and exonuclease activities in vitro. Acetylation occurs at four lysines located at the C terminus of Fen1, which is important for DNA binding. In this paper we show that Fen1 mutant proteins lacking the lysines at the C terminus have both reduced PCNA independent exonucleolytic and endonucleolytic activities. However, lysines at the C terminus are not required for PCNA stimulation of human Fen1. A double flap substrate was optimal for human Fen1 endonuclease and did not require the C-terminal lysines. Similarly, a one nucleotide 3'-overhang nick substrate was optimal for human Fen1 exonuclease and also did not require the C-terminal lysines. Finally, we found by an electromobility shift assay that human Fen1 had a different mode of binding with a double flap substrate containing a one nucleotide 3'-tail when compared to various other flap substrates. Taken together, our results confirm the double flap substrate as the likely in vivo intermediate for human Fen1 and that the C-terminal lysines are important for the endonuclease and exonuclease activities likely through DNA binding.     

Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation

Background

PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome.

Scope

This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution.

Conclusions

Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.     

Influence of chromatin and single strand binding proteins on the activity of an archaeal MCM

The mini-chromosome maintenance (MCM) complex is the presumptive replicative helicase in archaea and eukaryotes. In archaea, the MCM is a homo-multimer, in eukaryotes a heterohexamer composed of six related subunits, MCM 2-7. Biochemical studies using naked DNA templates have revealed that archaeal MCMs and a sub-complex of eukaryotic MCM 4, 6 and 7 have 3' to 5' helicase activity. Here, we investigate the influence of the major chromatin proteins, Alba and Sul7d, of Sulfolobus solfataricus (Sso) on the ability of the MCM complex to melt partial duplex DNA substrates. In addition, we test the effect of Sso SSB on MCM activity. We reveal that Alba represents a formidable barrier to MCM activity and further demonstrate that acetylation of Alba alleviates repression of MCM activity.