Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus

In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.     

Meiosis and small ubiquitin-related modifier (SUMO)-conjugating enzyme, Ubc9

In this review, we describe the role of a small ubiquitin-like protein modifier (SUMO)-conjugating protein, Ubc9, in synaptonemal complex formation during meiosis in a basidiomycete, Coprinus cinereus. Because its meiotic cell cycle is long and naturally synchronous, it is suitable for molecular biological, biochemical and genetic studies of meiotic prophase events. In yeast two-hybrid screening using the meiotic-specific cDNA library of C. cinereus, we found that the meiotic RecA homolog CcLim15 interacted with CcUbc9, CcTopII and CcPCNA. Moreover, both TopII and PCNA homologs were known as Ubc9 interactors and the targets of sumoylation. Immunocytochemistry demonstrates that CcUbc9, CcTopII and CcPCNA localize with CcLim15 in meiotic nuclei during leptotene to zygotene when synaptonemal complex is formed and when homologous chromosomes pair. We discuss the relationships between Lim15/Dmc1 (CcLim15), TopII (CcTopII), PCNA (CcPCNA) and CcUbc9, and subsequently, the role of sumoylation in the stages. We speculate that CcLim15 and CcTopII work in cohesion between homologous chromatins initially and then, in the process of the zygotene events, CcUbc9 works with factors including CcLim15 and CcTopII as an inhibitor of ubiquitin-mediated degradation and as a metabolic switch in the meiotic prophase cell cycle. After CcLim15-CcTopII dissociation, CcLim15 remains on the zygotene DNA and recruits CcUbc9, Rad54B, CcUbc9, Swi5-Sfr1, CcUbc9 and then CcPCNA in rotation on the C-terminus. Finally during zygotene, CcPCNA replaces CcLim15 on the DNA and the free-CcLim15 is probably ubiquitinated and disappears. CcPCNA may recruit the polymerase. The idea that CcUbc9 intervenes in every step by protecting CcLim15 and by switching several factors at the C-terminus of CcLim15 is likely. At the boundary of the zygotene and pachytene stages, CcPCNA would be sumoylated. CcUbc9 may also be involved with CcPCNA in the switch from the replicative polymerase being recruited at zygotene to the repair-type DNA polymerases being recruited at pachytene.     

Dissociation of DNA polymerase alpha-primase complex during meiosis in Coprinus cinereus.

Previously, the activity of DNA polymerase alpha was found in the meiotic prophase I including non-S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase alpha during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase alpha catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno-staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol alpha-primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol alpha-primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol alpha-primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase alpha-primase complex is altered during meiotic development.     

White-cap mutants and meiotic apoptosis in the basidiomycete Coprinus cinereus

Among many white-cap mutants of Coprinus cinereus, four distinct classes have been identified cytologically. Mutants of one class progress through meiosis normally but fail to sporulate; the defect is post-meiotic and it triggers apoptosis in the tetrad stage. Mutants of the other three classes have defects in meiotic prophase and these are: (1) those that assemble synaptonemal complexes (SCs) normally; (2) those that assemble axial elements (AEs) but not SCs; and (3) those that assemble neither AEs nor SCs even though the chromosomes are condensed and also paired. All three meiotic mutant classes arrest at meiotic metaphase I and the arrest triggers meiosis-specific apoptosis showing characteristic chromatin condensation, DNA fragmentation as shown by the TUNEL assay, cytoplasmic shrinkage, and finally total DNA degradation. Apoptosis is very cell-type specific; it occurs only in the basidia while the neighboring somatic cells are perfectly healthy and the mushroom continues to develop and mature with very few basidiospores produced. The meiotic apoptosis in C. cinereus is under strict cell cycle control rather than at any time after defect; apoptosis is triggered only after entry to meiotic metaphase. It is intriguing to note that C. cinereus has two checkpoints for arrest and entry to apoptosis: one is meiotic at the metaphase I spindle checkpoint regardless of the time of defects, and one is post-meiotic at the tetrad stage. This is in striking contrast to multiple checkpoint arrests and entries to meiotic apoptosis found in the mouse.     

Proliferating cell nuclear antigen from a basidiomycete, Coprinus cinereus. Alternative truncation and expression in meiosis.

The primary purpose of the present study was to investigate whether DNA replication at meiotic prophase also requires replication factors, especially proliferating cell nuclear antigen (PCNA). We cloned PCNA cDNAs (CoPCNA) from a cDNA library made from basidia of the basidiomycete, Coprinus cinereus. Interestingly, although CoPCNA is a single-copy gene in the genome, two different PCNA cDNA species were isolated using degenerate primers and a meiotic cDNA library, and were designated as CoPCNA-alpha and CoPCNA-beta. CoPCNA-beta was made by truncating at specific sites in CoPCNA-alpha mRNA, 5'-AAGAAGGAGAAG-3' and 5'-GAAGAGGAAGAA-3'. Both of these sequences were present in exon IV in the genomic sequence, and interestingly the former was the same as the inverse sequence of the latter. CoPCNA-alpha was 107 amino acids larger than human PCNA, and so the 107 amino-acid sequence was inserted in a loop, the so-called D2E2 loop, in human PCNA. Northern blotting analysis indicated that CoPCNA was expressed not only at premeiotic S but also at the meiotic prophase stages such as leptotene and early zygotene, just before and when karyogamy occurs and the homologous chromosomes pair. Western blotting analysis using anti-(CoPCNA-alpha) Ig revealed that at least two CoPCNA mRNAs before and after truncation were translated at the meiotic prophase as CoPCNA-alpha and CoPCNA-beta.     

An mre11 mutant of Coprinus cinereus has defects in meiotic chromosome pairing, condensation and synapsis

The rad11 gene of the basidiomycete Coprinus cinereus is required for the completion of meiosis and for survival after gamma irradiation. We have cloned the rad11 gene and shown that it is a homolog of MRE11, a gene required for meiosis and DNA repair in numerous organisms. The expression of C. cinereus mre11 is induced during prophase I of meiosis and following gamma irradiation. The gene encodes a predicted polypeptide of 731 amino acids, and the mre11-1 (rad11-1) mutation is a single base pair change that results in a stop codon after amino acid 315. The mre11-1 mutant shows enhanced sensitivity to ionizing radiation, but no enhanced sensitivity to UV radiation. It shows a delay in fruitbody formation and a reduction in the number of mushrooms formed per dikaryon. The mre11-1 mutant also has several meiotic defects. Pachytene chromatin condensation is disrupted, and although some meiotic cells appear to achieve metaphase I condensation, no further meiotic progression is observed. The mre11-1 mutant also fails to undergo proper chromosome synapsis; neither axial elements nor mature synaptonemal complexes are complete. Finally, meiotic homolog pairing is reduced in the mre11-1 mutant. Thus, in C. cinereus, Mre11 is required for meiotic DNA metabolism.     

Mammalian DNA ligases

DNA joining enzymes play an essential role in the maintenance of genomic integrity and stability. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Since DNA ligase II appears to be derived from DNA ligase III by a proteolytic mechanism, the three LIG genes can account for the four biochemically distinct DNA ligase activities, DNA ligases I, II, III and IV, that have been purified from mammalian cell extracts. It is probable that the specific cellular roles of these enzymes are determined by the proteins with which they interact. The specific involvement of DNA ligase I in DNA replication is mediated by the non-catalytic amino-terminal domain of this enzyme. Furthermore, DNA ligase I participates in DNA base excision repair as a component of a multiprotein complex. Two forms of DNA ligase III are produced by an alternative splicing mechanism. The ubiqitously expressed DNA ligase III-α forms a complex with the DNA single-strand break repair protein XRCC1. In contrast, DNA ligase III-β, which does not interact with XRCC1, is only expressed in male meiotic germ cells, suggesting a role for this isoform in meiotic recombination. At present, there is very little information about the cellular functions of DNA ligase IV.     

DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories

DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.     

Ca(2+)-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest

CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca2+ signal from the sperm that accelerates it. Here we visualised degradation of cyclin B1::GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca2+ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca2+ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca2+ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca2+ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca2+ directly stimulates destruction of CSF during mammalian fertilisation.     

Effects of cisplatin on expression of DNA ligases in MiaPaCa human pancreatic cancer cells

The effect of the broad-spectrum anticancer agent, cisplatin, on the expression of DNA ligase I in human pancreatic carcinoma MiaPaCa cells was examined in this study, since DNA ligase I is known to be involved in various DNA repair pathways. Upon exposure of MiaPaCa cells to cisplatin at near IC(50) value (2.5-5 microM), about 2-3-fold increase of DNA ligase I levels was observed within 24h, while levels of other DNA ligases (III and IV) remained unchanged or slightly decreased. The same fold-increase in DNA ligase I levels was also observed in MiaPaCa cells exposed to cytostatic concentrations, but not cytotoxic concentrations of cisplatin, which significantly reduced the number of cells. Flow cytometric analysis revealed that normal cell cycle progression was disrupted in the cells treated with cisplatin, resulting in an initial arrest of the cells in the S-phase, concomitant with a decrease of cells in G0/G1-phase. With time elapsing, the transition from S- to G2 + M-phase was observed, but further progression into G0/G1-phase was blocked. Overall, the increase of DNA ligase I expression seems to correlate well with the arrest of the cell cycle between the S- and G2-phases in response to cisplatin treatment. Interestingly, the cisplatin-induced DNA ligase I increase was abrogated by caffeine treatment in MiaPaCa cells, suggesting that caffeine sensitive kinases might be important mediators in the pathway, leading to the increase of DNA ligase I levels in response to cisplatin. We propose that the increase of DNA ligase I expression after exposure to cisplatin might be required for aiding the cells to recover from the damage by facilitating the repair process.     

An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.     

Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair

DNA ligase I belongs to a family of proteins that bind to proliferating cell nuclear antigen (PCNA) via a conserved 8-amino-acid motif [1]. Here we examine the biological significance of this interaction. Inactivation of the PCNA-binding site of DNA ligase I had no effect on its catalytic activity or its interaction with DNA polymerase beta. In contrast, the loss of PCNA binding severely compromised the ability of DNA ligase I to join Okazaki fragments. Thus, the interaction between PCNA and DNA ligase I is not only critical for the subnuclear targeting of the ligase, but also for coordination of the molecular transactions that occur during lagging-strand synthesis. A functional PCNA-binding site was also required for the ligase to complement hypersensitivity of the DNA ligase I mutant cell line 46BR.1G1 to monofunctional alkylating agents, indicating that a cytotoxic lesion is repaired by a PCNA-dependent DNA repair pathway. Extracts from 46BR.1G1 cells were defective in long-patch, but not short-patch, base-excision repair (BER). Our results show that the interaction between PCNA and DNA ligase I has a key role in long-patch BER and provide the first evidence for the biological significance of this repair mechanism.     

A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes

DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.     

Activation of mammalian DNA ligase I through phosphorylation by casein kinase II.

Mammalian DNA ligase I has been shown to be a phosphoprotein. Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein. Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity. Incubation of the full-length preparation from E. coli with purified casein kinase II (CKII) resulted in phosphorylation of the N-terminal region and was accompanied by activation of the DNA ligase. Of a variety of purified protein kinases tested, only CKII stimulated the activity of calf thymus DNA ligase I. Tryptic phosphopeptide analysis of DNA ligase I revealed that CKII specifically phosphorylated a major peptide also apparently phosphorylated in cells, implying that CKII is a protein kinase acting on DNA ligase I in the cell nucleus. These data suggest that DNA ligase I is negatively regulated by its N-terminal region and that this inhibition can be relieved by post-translational modification.     

Zyg-11 and cul-2 regulate progression through meiosis II and polarity establishment in C. elegans

The mechanisms that ensure coupling between meiotic cell cycle progression and subsequent developmental events, including specification of embryonic axes, are poorly understood. Here, we establish that zyg-11 and the cullin cul-2 promote the metaphase-to-anaphase transition and M phase exit at meiosis II in Caenorhabditis elegans. Our results indicate that ZYG-11 acts with a CUL-2-based E3 ligase that is essential at meiosis II and that functions redundantly with the anaphase-promoting complex/cyclosome at meiosis I. Our data also indicate that delayed M phase exit in zyg-11(RNAi) embryos is due to accumulation of the B type cyclin CYB-3. We demonstrate that PAR proteins and P granules become polarized in an inverted manner during the meiosis II delay resulting from zyg-11 or cul-2 inactivation, and that zyg-11 and cul-2 can regulate polarity establishment independently of a role in cell cycle progression. Furthermore, we find that microtubules appear dispensable for ectopic polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for AP polarity during the first mitotic cell cycle in wild-type embryos. Our findings suggest a model in which a CUL-2-based E3 ligase promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.     

Cell cycle-dependent phosphorylation of human DNA ligase I at the cyclin-dependent kinase sites

We have described previously that, during S-phase, human DNA ligase I is phosphorylated on Ser66, a casein kinase II site. Here we investigate the phosphorylation status of DNA ligase I during the cell cycle by gel shift analysis and electrospray mass spectrometry. We show that three residues (Ser51, Ser76, and Ser91), which are part of cyclin-dependent kinase sites, are phosphorylated in a cell cycle-dependent manner. Phosphorylation of Ser91 occurs at G1/S transition and depends on a cyclin binding site in the C-terminal part of the protein. This modification is required for the ensuing phosphorylation of Ser76 detectable in G2/M extracts. The substitution of serines at positions 51, 66, 76, and 91 with aspartic acid to mimic the phosphorylated enzyme hampers the association of DNA ligase I with the replication foci. We suggest that the phosphorylation of DNA ligase I and possibly other replicative enzymes is part of the mechanism that directs the disassembly of the replication machinery at the completion of S-phase.     

Purification of DNA ligases from mouse testis and their behavior during meiosis

Two types of DNA ligase, I and II, have been purified approximately 4,000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNA ligase I and II consisted of single polypeptides with molecular weights of 95,000 and 65,000, respectively, according to the estimation by SDS-polyacrylamide gel electrophoresis and the AMP-binding assay. Ligase activities were higher in premeiotic spermatogonia and spermatocytes than those in liver and bone marrow cells. Moreover, DNA ligase II showed rapid increase during meiotic prophase and a decrease in round spermatids. Since this behavior of DNA ligase II is consistent with that of m-rec and DNA polymerase beta, both of which have been shown to be involved in DNA recombination in meiotic cells, DNA ligase II might be an enzyme which works at the final step of meiotic recombination reaction.     

Identification of DNA replication and cell cycle proteins that interact with PCNA.

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.     

<Emphasis Type="Italic">Coprinus cinereus</Emphasis> Mer3 is required for synaptonemal complex formation during meiosis

Mer3 is an evolutionarily conserved DNA helicase that has crucial roles in meiotic recombination and crossover formation. We have identified the MER3 homolog in Coprinus cinereus (Ccmer3) and show that it is expressed in zygotene and pachytene meiocytes. Immunostaining analysis indicated that CcMer3 was localized on chromosomes at zygotene and pachytene and CcMer3 foci were more frequent on paired than unpaired chromosomes. We generated a C. cinereus mer3 mutant (#1) and found that it showed abnormal meiosis progression and underwent apoptosis after prophase I. Basidiospore production in #1 was reduced to 0.8% of the wild-type level; the spores showed slower germination at 25°C but were similar to the wild type at 37°C. Electron microscopic analysis of chromosome spreads revealed that axial elements were formed in the mutant but that synapsis was defective, resulting in a reduction in spore production. Our results demonstrate that CcMer3 is required for synaptonemal complex formation after axial elements align and is thus essential for homologous synapsis.