Phosphatidylglycerol is involved in the dimerization of photosystem II

Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1Delta3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1Delta3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two-dimensional crystals of the native dimer.     

Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro.

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.     

Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers.

The effects of short wave ultraviolet (UV)-induced DNA lesions on the catalytic activity of Drosophila melanogaster topoisomerase II were investigated. The presence of these photoproducts impaired the enzyme's ability to relax negatively supercoiled pBR322 plasmid molecules. As determined by DNA photolyase-catalyzed photoreactivation experiments, enzyme inhibition was due to the presence of cyclobutane pyrimidine dimers in the DNA. When 10-20 cyclobutane dimers were present per plasmid, the initial velocity of topoisomerase II-catalyzed DNA relaxation was inhibited approximately 50%. Decreased relaxation activity correlated with an inhibition of the DNA strand passage step of the enzyme's catalytic cycle. In contrast, UV-induced photoproducts did not alter the prestrand passage DNA cleavage/religation equilibrium of topoisomerase II either in the absence or presence of antineoplastic agents. Results of the present study demonstrate that the repair of cyclobutane pyrimidine dimers is important for the efficient catalytic function of topoisomerase II.     

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.     

Swiveling and decatenation of replicating simian virus 40 genomes in vivo.

We have found that type II topoisomerase inhibitors have two effects on replicating simian virus 40 genomes in vivo: production of catenated dimers and slowed replication of the last 5% of the genome. This suggests that type II topoisomerase simultaneously decatenates and facilitates replication fork movement at this stage of DNA replication. On the basis of this observation, a detailed model is proposed for the roles of topoisomerases I and II in simian virus 40 DNA replication.     

Identification of a potent decatenating enzyme from Escherichia coli

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.     

Functional and structural characterization of rhodopsin oligomers

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-beta-D-maltoside, n-tetradecyl-beta-D-maltoside, and n-hexadecyl-beta-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-beta-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-beta-D-maltoside and n-hexadecyl-beta-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-beta-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.     

Multicopy plasmid instability: the dimer catastrophe hypothesis

Multimer formation reduces plasmid copy number and is an established cause of segregational instability. Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations. We report here that dimer distribution is in fact heterogeneous in recombination-proficient strains. Most cells in the population contain only monomers; dimers are confined to a small sub-population from which plasmid-free daughters arise at high frequency. In a rec⁺ culture where 4% of pBR322 molecules are dimers, more than half are in dimer-only cells. We show that this situation is inevitable because dimers replicate at twice the rate of monomers. Runaway multimerization is avoided because dimer-containing cells grow more slowly than their monomer-containing counterparts. A computer simulation is used to show how dimers proliferate after formation by homologous recombination. The equilibrium concentration of dimers is proportional to the inter-plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.     

Monomer and multimer covalently closed circular forms of Rous sarcoma virus DNA.

Covalently closed circular molecules of viral DNA synthesized in virus-infected cells are composed mainly of monomers sedimenting at 22 to 27S in neutral sucrose gradients. These monomers are detected by annealing with complementary DNA or transfection assay. However, 11% of the infectious circles sediment faster than monomers. There is a peak at 32S which may correspond to dimer molecules. Traces of infectivity (about 3%) found between 32S and 65S suggest the presence of higher oligomers. In alkaline sucrose gradients, covalently closed monomers are found at 64 to 71S. Infectivity of these monomers is reduced by alkali treatment to less than one-tenth, and, perhaps for this reason, no infectious dimers or higher oligomers are observed. It has been shown that upon resedimentation the dimers of 95 can be separated from monomers and detected by hybridization.     

Sequences that promote formation of catenated intertwines during termination of DNA replication.

The normal sequence at which SV40 DNA replication terminates (TER) is unusual in that it promotes formation of catenated intertwines when two converging replication forks enter to complete replication (Weaver et al., 1985). Here we show that yeast centromeric sequences also exhibit this phenomenon. CEN3 caused accumulation of late replicating intermediates and catenated dimers in plasmids replicating in mammalian cells, but only when it was located in the termination region (180 degrees from ori), and only when cells were subjected to hypertonic shock to reduce topoisomerase II activity. Therefore, formation of catenated intertwines during termination of DNA replication was sequence dependent, suggesting that topoisomerase II acts behind replication forks in the termination region to remove intertwines generated by unwinding DNA rather than acting after replication is completed and catenates are formed. Under normal physiological conditions, CEN3 did not promote formation of catenated dimers in either mammalian or yeast cells. Therefore, CEN does not maintain association of sister chromatids during mitosis in yeast by introducing stable catenated intertwines during replication.     

Characterization of tubule and monomer derived from VP4 protein of infectious bursal disease virus

The VP4 protein of infectious bursal disease virus (IBDV) is a serine protease that processes the polyprotein for viral assembly. VP4 has been found to associate primarily with type II IBDV tubules that are 24 nm in diameter. In this study, a chimeric VP4, assigned as HS1VP4, was constructed with a VP4-autocleavage site inserted between the N-terminal His-tag and the VP4 sequence. The results showed that the VP4 forms tubules after the self-cleavage of HS1VP4 when expressed in Escherichia coli. Furthermore, a deletion of 28 amino acids at the C-terminus of VP4 resulted in monomers and dimers instead of tubule formation; mutants of S652A and K692A at active site destroyed the activity. The endopeptidase activity of these monomers and dimers was approximately 12.5 times higher than that of VP4 tubules. Additionally, the formation of tubules inhibited VP4 protease activity, as demonstrated through in vitro assays. The production and characterization of monomers or dimers that have greater endopeptidase activity and protease activity than tubules can provide further insight into VP4 tubule assembly and the regulation of VP4 activity in host cells; this insight will facilitate the development of new anti-IBDV strategies.     

Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo.

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.     

A biochemical analysis of topoisomerase II alpha and beta kinase activity found in HIV-1 infected cells and virus

Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (alpha) and topoisomerase II beta (beta). The alpha isoform is localized predominantly in the nucleus, while the beta isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II alpha, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II beta. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II alpha kinase activity after 8h p.i and topoisomerase beta kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II alpha and beta kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II alpha and beta, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II alpha. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II alpha and beta. The catalytic inhibitions by serine kinase inhibitors further suggest that the alpha and beta kinase activities associated with virus are distinctly different.     

Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.     

The termination region for SV40 DNA replication directs the mode of separation for the two sibling molecules

Separation of the two newly replicated chromosomes in simian virus 40 late replicating intermediates (RI*) occurred by two pathways. The parental DNA strands were completely unwound, releasing circular DNA monomers with a gap in the nascent strand (Form II*), or duplex DNA in the termination region was not unwound, resulting in formation of catenated dimers. Under optimal conditions, both products were transient intermediates in replication, although Form II* was predominant. However, in hypertonic medium both RI* and catenated dimers accumulated, and Form II* was not observed. Hypertonic medium appeared to inhibit both DNA unwinding in the termination region and separation of catenated dimers. When the size of the genome or the position of the origin of replication was changed, termination occurred at sites other than that of wild-type SV40. Neither catenated dimers nor RI* DNA accumulated at these sites. Instead, RI* separated into Form II*. Unwinding parental DNA was more difficult at some termination regions than others. Therefore, although completion of DNA replication does not require a unique termination sequence, this sequence can determine the mode of separation for sibling molecules.