Posttranscriptional modification of retroviral primers is required for late stages of DNA replication

During reverse transcription of retroviral RNA, synthesis of (−) strand DNA is primed by a cellular tRNA that anneals to an 18-nt primer binding site within the 5′ long terminal repeat. For (+) strand synthesis using a (−) strand DNA template linked to the tRNA primer, only the first 18 nt of tRNA are replicated to regenerate the primer binding site, creating the (+) strand strong stop DNA intermediate and providing a 3′ terminus capable of strand transfer and further elongation. On model HIV templates that approximate the (−) strand linked to natural modified or synthetic unmodified tRNA3Lys, we find that a (+) strand strong stop intermediate of the proper length is generated only on templates containing the natural, modified tRNA3Lys, suggesting that a posttranscriptional modification provides the termination signal. In the presence of a recipient template, synthesis after strand transfer occurs only from intermediates generated from templates containing modified tRNA3Lys. Reverse transcriptase from Moloney murine leukemia virus and avian myoblastosis virus shows the same requirement for a modified tRNA3Lys template. Because all retroviral tRNA primers contain the same 1-methyl-A58 modification, our results suggest that 1-methyl-A58 is generally required for termination of replication 18 nt into the tRNA sequence, generating the (+) strand intermediate, strand transfer, and subsequent synthesis of the entire (+) strand. The possibility that the host methyl transferase responsible for methylating A58 may provide a target for HIV chemotherapy is discussed.

Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+: poly(adenosine-diphosphate-d-ribosyl)-acceptor ADP-d-ribosyltransferase, EC 2.4.2.30] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP−/− mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by γ-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body γ-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP−/− cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

Equilibrium distributions of topological states in circular DNA: Interplay of supercoiling and knotting

Two variables define the topological state of closed double-stranded DNA: the knot type, K, and ΔLk, the linking number difference from relaxed DNA. The equilibrium distribution of probabilities of these states, P(ΔLk, K), is related to two conditional distributions: P(ΔLk|K), the distribution of ΔLk for a particular K, and P(K|ΔLk) and also to two simple distributions: P(ΔLk), the distribution of ΔLk irrespective of K, and P(K). We explored the relationships between these distributions. P(ΔLk, K), P(ΔLk), and P(K|ΔLk) were calculated from the simulated distributions of P(ΔLk|K) and of P(K). The calculated distributions agreed with previous experimental and theoretical results and greatly advanced on them. Our major focus was on P(K|ΔLk), the distribution of knot types for a particular value of ΔLk, which had not been evaluated previously. We found that unknotted circular DNA is not the most probable state beyond small values of ΔLk. Highly chiral knotted DNA has a lower free energy because it has less torsional deformation. Surprisingly, even at |ΔLk| > 12, only one or two knot types dominate the P(K|ΔLk) distribution despite the huge number of knots of comparable complexity. A large fraction of the knots found belong to the small family of torus knots. The relationship between supercoiling and knotting in vivo is discussed.

Femtosecond cluster studies of the solvated 7-azaindole excited state double-proton transfer

Presented here are femtosecond pump-probe studies on the water-solvated 7-azaindole dimer, a model DNA base pair. In particular, studies are presented that further elucidate the nature of the reactive and nonreactive dimers and also provide new insights establishing that the excited state double-proton transfer in the dimer occurs in a stepwise rather than a concerted manner. A major question addressed is whether the incorporation of a water molecule with the dimer results in the formation of species that are unable to undergo excited state double-proton transfer, as suggested by a recent study reported in the literature [Nakajima, A., Hirano, M., Hasumi, R., Kaya, K., Watanabe, H., Carter, C. C., Williamson, J. M. & Miller, T. (1997) J. Phys. Chem. 101, 392–398]. In contrast to this earlier work, our present findings reveal that both reactive and nonreactive dimers can coexist in the molecular beam under the same experimental conditions and definitively show that the clustering of water does not induce the formation of the nonreactive dimer. Rather, when present with a species already determined to be a nonreactive dimer, the addition of water can actually facilitate the occurrence of the proton transfer reaction. Furthermore, on attaining a critical hydration number, the data for the nonreactive dimer suggest a solvation-induced conformational structure change leading to proton transfer on the photoexcited half of the 7-azaindole dimer.

Topoisomerase II drives DNA transport by hydrolyzing one ATP

DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport. Current models for the enzyme’s mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction. Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner. This result is incongruous with the models based on the nonhydrolyzable ATP analog data. Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport. These results indicate that important features of this enzyme’s mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.

Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) α enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCRα enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCRα enhancer.

Construction of a Z-DNA-specific restriction endonuclease

Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Zα) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADAR1), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Zα nuclease, which is a chimera of Zα and FokI nuclease. Purified Zα nuclease cleaves negatively supercoiled plasmids only when they contain a Z-DNA forming insert, such as (dC-dG)13. The precise location of the cleavage sites was determined by primer extension. Cutting has been mapped to the edge of the B-Z junction, suggesting that Zα nuclease binds within the Z-DNA insert, but cleaves in the nearby B-DNA, by using a mechanism similar to type IIs restriction enzymes. These data show that Zα binds Z-DNA in an environment similar to that in a cell. Zα nuclease, a structure-specific restriction enzyme, may be a useful tool for further study of the biological role of Z-DNA.

Physical interaction between components of DNA mismatch repair and nucleotide excision repair

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as “bait,” and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Identification and characterization of a single-stranded DNA-binding protein from the archaeon Methanococcus jannaschii

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria and eukarya. We report here the identification and characterization of the SSB of an archaeon, Methanococcus jannaschii. The M. jannaschii SSB (mjaSSB) has significant amino acid sequence similarity to the eukaryotic SSB, replication protein A (RPA), and contains four tandem repeats of the core single-stranded DNA (ssDNA) binding domain originally defined by structural studies of RPA. Homologous SSBs are encoded by the genomes of other archaeal species, including Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus. The purified mjaSSB binds to ssDNA with high affinity and selectivity. The apparent association constant for binding to ssDNA is similar to that of RPA under comparable experimental conditions, and the affinity for ssDNA exceeds that for double-stranded DNA by at least two orders of magnitude. The binding site size for mjaSSB is ≈20 nucleotides. Given that RPA is related to mjaSSB at the sequence level and to Escherichia coli SSB at the structural level, we conclude that the SSBs of archaea, eukarya, and bacteria share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

Double-stranded RNA induces mRNA degradation in Trypanosoma brucei

Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the α-tubulin mRNA 5′ untranslated region (5′ UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic α-tubulin 5′ UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of α-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to α-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.

Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage

The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase. However, these modifications alone may not account in full for p53 stabilization. p53's stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner. This phosphorylation is independent of p53 and the DNA-dependent protein kinase. Furthermore, MDM2 is directly phosphorylated by ATM in vitro. These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.

Topoisomerase II-mediated site-directed alkylation of DNA by psorospermin and its use in mapping other topoisomerase II poison binding sites

Psorospermin is a plant natural product that shows significant in vivo activity against P388 mouse leukemia. The molecular basis for this selectivity is unknown, although psorospermin has been demonstrated to intercalate into DNA and alkylate N7 of guanine. Significantly, the alkylation reactivity of psorospermin at specific sites on DNA increased 25-fold in the presence of topoisomerase II. In addition, psorospermin trapped the topoisomerase II-cleaved complex formation at the same site. These results imply that the efficacy of psorospermin is related to its interaction with the topoisomerase II–DNA complex. Because thermal treatment of (N7 guanine)–DNA adducts leads to DNA strand breakage, we were able to determine the site of alkylation of psorospermin within the topoisomerase II gate site and infer that intercalation takes place at the gate site between base pairs at the +1 and +2 positions. These results provide not only additional mechanistic information on the mode of action of the anticancer agent psorospermin but also structural insights into the design of an additional class of topoisomerase II poisons. Because the alkylation site for psorospermin in the presence of topoisomerase II can be assigned unambiguously and the intercalation site inferred, this drug is a useful probe for other topoisomerase poisons where the sites for interaction are less well defined.

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.

Plasmon analyses of Triticum (wheat) and Aegilops: PCR–single-strand conformational polymorphism (PCR-SSCP) analyses of organellar DNAs

To investigate phylogenetic relationships among plasmons in Triticum and Aegilops, PCR–single-strand conformational polymorphism (PCR-SSCP) analyses were made of 14.0-kb chloroplast (ct) and 13.7-kb mitochondrial (mt)DNA regions that were isolated from 46 alloplasmic wheat lines and one euplasmic line. These plasmons represent 31 species of the two genera. The ct and mtDNA regions included 10 and 9 structural genes, respectively. A total of 177 bands were detected, of which 40.6% were variable. The proportion of variable bands in ctDNA (51.1%) was higher than that of mtDNA (28.9%). The phylogenetic trees of plasmons, derived by two different models, indicate a common picture of plasmon divergence in the two genera and suggest three major groups of plasmons (Einkorn, Triticum, and Aegilops). Because of uniparental plasmon transmission, the maternal parents of all but one polyploid species were identified. Only one Aegilops species, Ae. speltoides, was included in the Triticum group, suggesting that this species is the plasmon and B and G genome donor of all polyploid wheats. ctDNA variations were more intimately correlated with vegetative characters, whereas mtDNA variations were more closely correlated with reproductive characters. Plasmon divergence among the diploids of the two genera largely paralleled genome divergence. The relative times of origin of the polyploid species were inferred from genetic distances from their putative maternal parents.