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.     

The DNA polymerase III holoenzyme: an asymmetric dimeric replicative complex with leading and lagging strand polymerases.

The DNA Polymerase III holoenzyme forms initiation complexes on primed DNA in an ATP-dependent reaction. We demonstrate that the nonhydrolyzable ATP analog, ATP gamma S, supports the formation of an isolable leading strand complex that loads and replicates the lagging strand only in the presence of ATP, beta, and the single-stranded DNA binding protein. The single endogenous DnaX complex within DNA polymerase III holoenzyme assembles beta onto both the leading and lagging strand polymerases by an ordered mechanism. The dimeric replication complex disassembles in the opposite order from which it assembled. Upon ATP gamma S-induced dissociation, the leading strand polymerase is refractory to disassembly allowing cycling to occur exclusively on the lagging strand. These results establish holoenzyme as an intrinsic asymmetric dimer with distinguishable leading and lagging strand polymerases.     

Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA.

Five monoclonal antibodies raised against rat PCNA cross-reacted with a similar protein in the fission yeast Schizosaccharomyces pombe. One of these was used to screen an S.pombe cDNA expression library. An incomplete cDNA was isolated and used to screen a genomic library, identifying a single gene, designated pcn1+ (proliferating cell nuclear antigen). The gene encodes a protein of 260 amino acids, with a deduced sequence 52% identical to human and rat PCNAs, which are 98.5% identical to each other. The budding yeast PCNA homologue POL30 is only 35% identical to the human and rat proteins. Pcn1 has a region near the C-terminus of particularly high homology to higher eukaryotic PCNA proteins. pcn1+ is essential for viability and delta pcn1 cells undergo aberrant DNA replication before cell cycle arrest. Overproduction of the protein leads to cell cycle delay in G2. Disruption of pcn1+ is complemented by the human PCNA gene, demonstrating that these genes are functional homologues.     

Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (～0.2 to 0.6 kb) were significantly shorter than leading strand products (～2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.     

Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.     

Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase

The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.     

Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta

The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus. Specifically, we show that deletion of PIF1 suppresses the lethality of a DNA2-null mutant. The pif1delta dna2delta strain remains methylmethane sulfonate sensitive and temperature sensitive; however, these phenotypes can be suppressed by further deletion of a subunit of pol delta, POL32. Deletion of PIF1 also suppresses the cold-sensitive lethality and hydroxyurea sensitivity of the pol32delta strain. Dna2 is thought to function by cleaving long flaps that arise during OFP due to excessive strand displacement by pol delta and/or by an as yet unidentified helicase. Thus, suppression of dna2delta can be rationalized if deletion of POL32 and/or PIF1 results in a reduction in long flaps that require Dna2 for processing. We further show that deletion of DNA2 suppresses the long-telomere phenotype and the high rate of formation of gross chromosomal rearrangements in pif1Delta mutants, suggesting a role for Dna2 in telomere elongation in the absence of Pif1.     

Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented.     

Requirements for synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and gene 4 protein of bacteriophage T7.

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze DNA synthesis on duplex DNA templates. Synthesis is initiated at nicks in the DNA template, and this leading strand synthesis results in displacement of one of the parental strands. In the presence of ribonucleoside 5'-triphosphates the gene 4 protein catalyzes the synthesis of oligoribonucleotide primers on the displaced single strand, and their extension by T7 dna polymerase accounts for lagging strand synthesis. Since all the oligoribonucleotide primers bear adenosine 5'-triphosphate residues at their 5' termini, [gamma 32P]ATP is incorporated specifically into the product molecule, thus providing a rapid and sensitive assay for the synthesis of the RNA primers. Both primer synthesis and DNA synthesis are stimulated 3- to 5-fold by the presence of either Escherichia coli or T7 helix-destabilizing protein (DNA binding protein). ATP and CTP together fully satisfy the requirement for rNTPs and provide maximum synthesis of primers and DNA. Provided that T7 DNA polymerase is present, RNA-primed DNA synthesis occurs on either duplex or single-stranded DNA templates and to equal extents on either strand of T7 DNA. No primer-directed DNA synthesis occurs on poly(dT) or poly(dG) templates, indicating that synthesis of primers is template-directed.     

DNA primase isolated from the yeast DNA primase-DNA polymerase complex. Immunoaffinity purification and analysis of RNA primer synthesis

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.     

Mutator phenotype resulting from DNA polymerase IV overproduction in Escherichia coli: preferential mutagenesis on the lagging strand

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.     

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.     

Control over the onset of DNA synthesis in fission yeast

The fission yeast Schizosaccharomyces pombe has been used to identify gene functions required for the cell to become committed to the mitotic cell cycle and to initiate the processes leading to chromosome replication in S-phase. Two gene functions cdc2 and cdc10 must be executed for the cell to traverse 'start' and proceed from G1 into S-phase. Before the completion of these two functions the cell is in an uncommitted state and can undergo alternative developmental fates such as conjugation. A third gene, suc1, has also been identified whose product may interact directly with that of cdc2 at 'start'. The molecular functions of the genes involved in the completion of 'start' have been investigated. The cdc2 gene has been shown to be a protein kinase, suggesting that phosphorylation may be involved in the control over the transition from G1 into S-phase. The biochemical functions of the cdc10 and suc1 gene products have not yet been elucidated. A control at 'start' has also been shown to exist in the budding yeast Saccharomyces cerevisiae. Traverse of 'start' requires the execution of the CDC28 gene function. The cdc2 and CDC28 gene products (lower-case letters represent genes of Schizosaccharomyces pombe, and capital letters genes of Saccharomyces cerevisiae) are functionally homologous, suggesting that the processes involved in traverse of 'start' are highly conserved. An analogous control may also exist in the G1 period of mammalian cells, suggesting that the 'start' control step, after which cells become committed to the mitotic cell cycle, may have been conserved through evolution.     

Functional interplay of DnaE polymerase,DnaG primase and DnaC helicase within a ternary complex,and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis

Bacillus subtilis has two replicative DNA polymerases. PolC is a processive high-fidelity replicative polymerase, while the error-prone DnaE_Bs extends RNA primers before hand-off to PolC at the lagging strand. We show that DnaE_Bs interacts with the replicative helicase DnaC and primase DnaG in a ternary complex. We characterize their activities and analyse the functional significance of their interactions using primase, helicase and primer extension assays, and a ‘stripped down’ reconstituted coupled assay to investigate the coordinated displacement of the parental duplex DNA at a replication fork, synthesis of RNA primers along the lagging strand and hand-off to DnaE_Bs. The DnaG–DnaE_Bs hand-off takes place after de novo polymerization of only two ribonucleotides by DnaG, and does not require other replication proteins. Furthermore, the fidelity of DnaE_Bs is improved by DnaC and DnaG, likely via allosteric effects induced by direct protein–protein interactions that lower the efficiency of nucleotide mis-incorporations and/or the efficiency of extension of mis-aligned primers in the catalytic site of DnaE_Bs. We conclude that de novo RNA primer synthesis by DnaG and initial primer extension by DnaE_Bs are carried out by a lagging strand–specific subcomplex comprising DnaG, DnaE_Bs and DnaC, which stimulates chromosomal replication with enhanced fidelity.     

Genetic analysis of the DnaA-dependent priming in the initiation of lagging strand DNA synthesis of plasmid pUB110 in Bacillus subtilis

Abstract Derivatives of Bacillus subtilis plasmid pUB110 lacking the major lagging strand replication origin ( sso U⁻) accumulate intracellular single-strand circular (SS(c)) DNA intermediates and are unable to propagate in dna B and dna D hosts. DnaA-dependent priming requires a DnaA box in a stable hairpin form; a higher copy number of a DnaA box is not sufficient as a signal for the conversion of the SS(c) into its dsDNA form. The introduction into the plasmid of a hairpin structure, whose stem carries a DnaA box, mediates conversion of SS(c) into dsDNA and makes plasmid replication independent of the B. subtilis dna B function. This conversion signal has been termed sso A.     

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.     

Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.     

Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 ofSchizosaccharomyces pombe was expressed inEscherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with³²P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutantrpb2 alleles,rpb2 ^E357A andrpb2 ^H3a6L , each carrying a single amino acid substitution at the site correponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutantrpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutantrpb2 alleles were expressed in anrpb2-disruptedS. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.     

Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex.

Yeast DNA primase and DNA polymerase I can be purified by immunoaffinity chromatography as a multipeptide complex which can then be resolved into its functional components and further reassembled in vitro. Isolated DNA primase synthesizes oligonucleotides of a preferred length of 9-10 nucleotides and multiples thereof on a poly(dT) template. In vitro reconstitution of the DNA primase:DNA polymerase complex allows the synthesis of long DNA chains covalently linked to RNA initiators shorter than those synthesized by DNA primase alone. The SS (single-stranded) circular DNA of phage M13mp9 can also be replicated by the DNA primase:DNA polymerase complex. Priming by DNA primase occurs at multiple sites and the initiators are utilized by the DNA polymerase moiety of the complex, so that almost all the SS template is converted into duplex form. The rate of DNA synthesis catalyzed by isolated yeast DNA polymerase I on the M13mp9 template is not constant and is characterized by distinct pausing sites, which partly correlate with secondary structures on the template DNA. Thus, replication of M13mp9 SS DNA with the native primase:polymerase complex gives rise to a series of DNA chains with significantly uniform termini specified by the primase start sites and the polymerase stop sites.