Kinetics of reaction of DNA-bound Fe(III)bleomycin with ascorbate: interplay of specific and non-specific binding

The aerobic redox reaction of Fe(III)bleomycin (Blm) and ascorbate was examined in the absence of DNA and in the presence of 7.5 and 25 calf thymus DNA base pairs per-drug molecule, in order to investigate the effect of DNA binding on the properties of FeBlm activation and DNA strand cleavage. Under these successive conditions, the rate of initial reduction of Fe(III)Blm became progressively slower and biphasic. Using 7.5 base pairs per-molecule of FeBlm, 2-3 times as much drug reacted in the faster step as with the larger DNA to drug ratio. In each case, the more rapid process was identified with the reaction of high spin Fe(III)Blm-DNA. With the smaller ratio, dioxygen consumption, formation of HO(2)-Fe(III)Blm-DNA, and production of DNA strand breaks as measured by the formation of base propenal were largely rate limited by the initial reaction of ascorbate with Fe(III)Blm-DNA. After a burst of reaction with the larger ratio of base pairs to Fe(III)Blm, a small fraction of the total Fe(III)Blm, representing high spin Fe(III)Blm, entered a steady state as HO(2)-Fe(III)Blm-DNA. Thereafter, reaction of dioxygen and base propenal formation occurred slowly with similar first-order rate kinetics. In order to explain these results, it is hypothesized that the metal domain-linker of Fe(III)Blm adopts two conformations with respect to DNA. One, at specific binding sites, is relatively unreactive with ascorbate. The other, present at non-specific sites as HPO(4)-Fe(III)Blm, is readily reactive with ascorbate to generate HO(2)-Fe(III)Blm-DNA. At the larger base pair to drug ratio, movement of Fe(III)Blm between specific and non-specific sites to generate HO(2)-Fe(III)Blm is a necessary part of the mechanism of strand scission.     

Interactions of iron bleomycin, phosphate or cyanide, and DNA: sequence-dependent conformations and reactions

The hypothesis was investigated that axial ligands bound to Fe(III)-bleomycin [Fe(III)Blm] are destabilized at specific 5'-guanine-pyrimidine-3' binding sites but are stable at nonselective dinucleotides. DNA oligomers and calf-thymus DNA were used in reactions with L-Fe(III)Blm, where phosphate and cyanide served as examples of large and small ligands (L). Both ligands underwent dissociation when L-Fe(III)Blm was bound to d(GGAAGCTTCC)2 (I) but not d(GGAAATTTCCC)2 (II) and at large ratios of calf-thymus DNA to drug. Fe(III)Blm is high spin in 20 mM phosphate buffer, signifying the presence of a phosphate adduct. In the titration of HPO4-Fe(III)Blm with calf-thymus DNA, a large excess of DNA was needed to reach the low-spin state, consistent with an equilibrium competition between phosphate and DNA for Fe(III)Blm. Equilibrium constants for binding Fe(III)Blm and CN-Fe(III)Blm to calf-thymus DNA (6.8x10(5) M(-1) and 5.9x10(4) M(-1), respectively, in HEPES buffer at 25 degrees C and pH 7.4) showed that the CN- ligand also reduced the affinity of DNA for the drug. The kinetics of dissociation of CN- from CN-Fe(III)Blm-DNA were slow and first order in bound drug. The reversible nature of these dissociation reactions was shown using 1H NMR spectroscopy of Fe(III)Blm-I in the absence and presence of large excesses of CN- or phosphate. The results are discussed in terms of a two-state hypothesis for the binding of L-Fe(III)Blm to specific and nonspecific dinucleotides. It is proposed that steric restrictions at specific sites inhibit binding of these ligands.     

Raman spectroscopy of an O(2)-Co(II)bleomycin-calf thymus DNA adduct: alternate polymer conformations.

Bleomycin (Blm) is an antitumor agent which binds to specific sequences of DNA and as HO(2)-Fe(III)Blm causes single and double strand cleavage. In the present investigation, binding of O(2)-Co(II)Blm to a native DNA polymer, calf thymus DNA, was examined using conventional Raman spectroscopy. O(2)-Co(II)Blm is a model for O(2)-Fe(II)Blm, the direct precursor of HO(2)-Fe(III)Blm. Although the DNA polymer retained a predominant B-form structure, Raman spectral evidence was obtained for localized structural changes to A, C and Z-DNA forms. The presence of these alternate DNA forms within B-DNA implied the presence of B/A, B/C and B/Z junctions. The observed changes in DNA secondary structure were attributed to perturbation of structural water resulting from binding of O(2)-Co(II)Blm within the minor groove.     

The effect of bleomycin on DNA synthesis in human cells: evidence for grouping of initiation of replicons in the S-period

The effect of bleomycin (Blm) on DNA synthesis has been studied in a synchronous culture of human embryonic lung cells. The data obtained suggest that in the Blm presence in a medium (20 micrograms/ml) DNA synthesis initiation in new replicons is suppressed. The Blm action at different S-phase intervals has been shown to inhibit DNA synthesis unequally. Four discrete time intervals have been singled out in the course of the 10-hr S-phase in which a grouped initiation of replicon portions can be supposed. Together with the data on DNA replication in large-size replicon units (50-500 microns), the obtained results account well for the uneven DNA synthesis in S-phase, manifested by 3 or 4 peaks of [3H]-thymidine incorporation in pulse-labelled cells.     

Structure of the Blm10-20 S proteasome complex by cryo-electron microscopy. Insights into the mechanism of activation of mature yeast proteasomes

The 20 S proteasome is regulated at multiple levels including association with endogenous activators. Two activators have been described for the yeast 20 S proteasome: the 19 S regulatory particle and the Blm10 protein. The sequence of Blm10 is 20% identical to the mammalian PA200 protein. Recent studies have shown that the sequences of Blm10 and PA200 each contain multiple HEAT-repeats and that each binds to the ends of mature proteasomes, suggesting a common structural and biochemical function. In order to advance structural studies, we have developed an efficient purification method that produces high yields of stoichiometric Blm10-mature yeast 20 S proteasome complexes and we constructed a three-dimensional (3D) model of the Blm10-20 S complex from cryo-electron microscopy images. This reconstruction shows that Blm10 binds in a defined orientation to both ends of the 20 S particle and contacts all the proteasome alpha subunits. Blm10 displays the solenoid folding predicted by the presence of multiple HEAT-like repeats and the axial gates on the alpha rings of the proteasome appear to be open in the complex. We also performed a genetic analysis in an effort to identify the physiological role of Blm10. These experiments, however, did not reveal a robust phenotype upon gene deletion, overexpression, or in a screen for synthetic effects. This leaves the physiological role of Blm10 unresolved, but challenges earlier findings of a role in DNA repair.     

Structures of HO(2)-Co(III)bleomycin A(2) bound to d(GAGCTC)(2) and d(GGAAGCTTCC)(2): structure-reactivity relationships of Co and Fe bleomycins

HO(2)-Co(III)bleomycin is a model for HO(2)-Fe(III)bleomycin, which initiates single and double strand cleavage of DNA. In order to enlarge the understanding of its structure and reactivity, three-dimensional structures of HO(2)-Co(III)bleomycin bound to two DNA oligomers, d(GAGCTC)(2) (I) and d(GGAAGCTTCC)(2) (II), that have 5'-GC-3' binding sites, have been determined by nuclear magnetic resonance (NMR) methods. Besides previously recognized determinants of binding selectivity, a probable hydrogen bond was detected between the pyrimidinyl acetamido NH(2) and the carbonyl of cytosine base paired to G at the recognition site. Another hydrogen bond between the NH of the dimethylsulfonium R group and N7 of guanine opposite cytosine at the GC site may contribute to specification of the pyrimidine. Substitution of G with inosine shifted HO(2)-Co(III)Blm A(2)[bond]I and Fe(III)Blm[bond]I into fast exchange on the NMR time scale, supporting the role of the 2-amino group in site specification for each molecule. The conformationally stable metal-domain linker established a close-packed adduct with the minor groove in which the hydroperoxide ligand occupies a sterically constrained pocket that is isolated from the solvent. The hydroperoxide group is directed toward one of the two cytosine H4' hydrogens but is sterically blocked from access to the other by the drug. These findings enlarge the structural understanding of selective binding of Co(III)/Fe(III)Blm species at G-pyrimidine sites. They also rationalize the instability of a number of ligands bound to Co(III)/Fe(III)Blm at specific binding sequences and the relative unreactivity of Fe(III)Blm[bond]I with ascorbate as well as its lack of interaction with spin labels.     

Comparative binding properties of metallobleomycins with DNA 10-mers.

Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2), ZnBlm A(2), Cu(II)Blm A(2), Fe(III)Blm A(2), and HO(2)-Co(III)Blm A(2). Generally, the metallobleomycins were bound with a modestly higher affinity to I. One-dimensional (1)H NMR spectra of the imino proton region of I in the presence of this series of compounds revealed that Blm and Zn- and CuBlm bind in fast exchange on the NMR time scale, while the Fe and Co complexes bind in slow exchange. Blm, ZnBlm, and Cu(II)Blm caused little perturbation of the UV circular dichroism spectrum of I or II. In contrast, Fe(III)Blm and HO(2)-Co(III)Blm induced hypochromic effects in the CD spectrum of I and altered the spectrum of II to a smaller extent. On the basis of these results, the DNA binding structures and properties of Blm A(2), ZnBlm A(2), and CuBlm A(2) differ substantially from those of Fe(III)Blm A(2) and HO(2)-Co(III)Blm A(2).     

Essential functions of C terminus of Drosophila Topoisomerase IIIα in double holliday junction dissolution

Topoisomerase IIIα (Top3α) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans, along with Blm and Rmi1/2. This important anti-recombinogenic function cannot be performed by Top3β, the other type IA topoisomerase present in metazoans. The two share a catalytic core but diverge in their tail regions. To understand this difference in function, we investigated the role of the unique C terminus of Top3α. The Drosophila C terminus contains an insert region not conserved among metazoans. This insert contributes an independent interaction with Blm, which may account for the absence of Rmi1 in Drosophila. Mutant Top3α lacking this insert maintains the ability to perform dHJ dissolution but only partially rescues a top3α null fly line, indicating an in vivo role for the insert. Truncation of the C terminus has a minimal effect on the type IA relaxation activity of Top3α; however, dHJ dissolution is greatly reduced. The Top3α C terminus was found to strongly interact with both Blm and DNA, which are critical to the dissolution reaction; these interactions are greatly reduced in the truncated enzyme. The truncation mutant also cannot rescue the viability of top3α null flies, indicating an essential in vivo role. Our data therefore suggest that the Top3α C terminus has an important role in dHJ dissolution (by providing an interaction interface for Blm and DNA) and an essential function in vivo.     

Reaction of nitric oxide with iron bleomycin bound to DNA: properties of the nitrosyl adduct and its reaction with O2

During the ESR spectroscopic titration of nitrosyl-iron bleomycin, ON---Fe(II)Blm, with DNA, its metal domain undergoes a change in environment as the DNA base pair to drug ratio increases to 50 to 1. The ¹⁵N---O stretching frequency of ON---Fe(II)Blm occurs at 1589 cm⁻¹, similar to that for nitrosyl hemoglobin and myoglobin. Upon addition of DNA (3 base pairs per drug molecule), this vibration is substantially broadened. Injection of O₂ into a solution of ON---Fe(II)BlmDNA converts the ESR signal of the nitrosyl species to low spin Fe(III) BlmDNA. NO is largely oxidized to NO₂⁻. The combination of these products suggests that the initial reaction of ON---Fe(II)Blm with O₂ generates Fe(III)Blm and peroxynitrite, O₂NO⁻. If peroxynitrite is formed in the reaction, it does not cause detectable DNA damage. The structural integrity of a supercoiled DNA plasmid, pBR322, is not compromised and no base propenals are produced during this reaction.     

Blm10 facilitates nuclear import of proteasome core particles

Short‐lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT‐like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP‐CP assemblies are primarily nuclear and promote cell division. During quiescence, RP‐CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10‐bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG‐rich nucleoporins and is dissociated from the CP by Ran‐GTP. Thus, Blm10 represents the first CP‐dedicated nuclear import receptor in yeast.     

Drosophila bloom helicase maintains genome integrity by inhibiting recombination between divergent DNA sequences

DNA double strand breaks (DSB) can be repaired either via a sequence independent joining of DNA ends or via homologous recombination. We established a detection system in Drosophila melanogaster to investigate the impact of sequence constraints on the usage of the homology based DSB repair via single strand annealing (SSA), which leads to recombination between direct repeats with concomitant loss of one repeat copy. First of all, we find the SSA frequency to be inversely proportional to the spacer length between the repeats, for spacers up to 2.4 kb in length. We further show that SSA between divergent repeats (homeologous SSA) is suppressed in cell cultures and in vivo in a sensitive manner, recognizing sequence divergences smaller than 0.5%. Finally, we demonstrate that the suppression of homeologous SSA depends on the Bloom helicase (Blm), encoded by the Drosophila gene mus309. Suppression of homeologous recombination is a novel function of Blm in ensuring genomic integrity, not described to date in mammalian systems. Unexpectedly, distinct from its function in Saccharomyces cerevisiae, the mismatch repair factor Msh2 encoded by spel1 does not suppress homeologous SSA in Drosophila.     

Studies on the chemical reactivity of copper bleomycin

The copper(II) complex of the clinically used antitumor agent bleomycin (Blm) has cytotoxic as well as antitumor properties. To understand the relationship of the bleomycin ligand, copper bleomycin, and other possible metal complexes of this agent, kinetic studies of the formation of Cu(II)Blm, ligand substitution reactions of CuBlm with ethylenediaminetetraaletic acid, and the redox reaction of CuBlm with thiols have been completed and interpreted along with previous studies of the thermodynamic stability of Cu2+ with bleomycin. Cu(II)Bm is found to be kinetically and thermodynamically stable in ligand substitution processes and is only slowly reduced and dissociated by sulfhydryl reagents. The rate constant of reduction of the complex by 2-mercaptoethanol (2-ME) at pH 7.4 and 25 degrees C is 9.5 X 10(-3) M-1 sec-1, explaining the inhibition of Fe2+-dependent strand scission of DNA by Cu2+ in the presence of 2-ME. CuBlm forms in preference to Fe(II)Blm and cannot be reduced and dissociated rapidly enough by thiols to liberate Blm and form the reactive iron complex. In agreement with the observed chemical stability of CuBlm, it is also shown that the complex is stable in human plasma and in the presence of Ehrlich cells suspended in ascites fluid. Interestingly, little CuBlm enters these cells to carry out cytotoxic reactions. Finally, it is shown that both Cu2+ and Zn2+, at equivalent concentrations to Fe2+, effectively inhibit the strand scission of DNA by Fe(II)Blm plus oxygen. However, at substoichiometric amounts of Cu2+, the ferroxidase activity of Blm enables the drug to remain effective in the strand-scission reaction, despite the lowered Cu-free Blm/Fe2+ ratio. These results are discussed in light of the proposed mechanism of action of bleomycin.     

Chromosome instability and tumor predisposition inversely correlate with BLM protein levels

Independent mouse models for Bloom syndrome (BS) exist, each thought to disrupt Blm gene function. However, animals bearing these alleles exhibit distinct phenotypes. Blm(tm1Ches) and Blm(tm1Grdn) homozygous mutant animals exhibit embryonic lethality while in another, Blm(tm3Brd), homozygosity yields viable, fertile animals with a cancer predisposition. Further characterization reveals the Blm(tm3Brd) allele to be a hypomorph, producing a diminished quantity of normal mRNA and protein. The Blm(tm3Brd) allele produces sufficient normal protein to rescue Blm(tm1Ches) lethality. Evaluation of viable animals reveals an inverse correlation between the quantity of Blm protein and the level of chromosome instability and a similar genotypic relationship for tumor predisposition indicating that Blm protein is rate limiting for maintaining genomic stability and the avoidance of tumors.     

The Bloom's syndrome helicase is critical for development and function of the alphabeta T-cell lineage

Bloom's syndrome is a genetic disorder characterized by increased incidence of cancer and an immunodeficiency of unknown origin. The BLM gene mutated in Bloom's syndrome encodes a DNA helicase involved in the maintenance of genomic integrity. To explore the role of BLM in the immune system, we ablated murine Blm in the T-cell lineage. In the absence of Blm, thymocytes were severely reduced in numbers and displayed a developmental block at the beta-selection checkpoint that was partially p53 dependent. Blm-deficient thymocytes rearranged their T-cell receptor (TCR) beta genes normally yet failed to survive and proliferate in response to pre-TCR signaling. Furthermore, peripheral T cells were reduced in numbers, manifested defective homeostatic and TCR-induced proliferation, and produced extensive chromosomal damage. Finally, CD4(+) and CD8(+) T-cell responses were impaired upon antigen challenge. Thus, by ensuring genomic stability, Blm serves a vital role for development, maintenance, and function of T lymphocytes, suggesting a basis for the immune deficiency in Bloom's syndrome.     

Blm10 protein promotes proteasomal substrate turnover by an active gating mechanism

For optimal proteolytic function, the central core of the proteasome (core particle (CP) or 20S) has to associate with activators. We investigated the impact of the yeast activator Blm10 on proteasomal peptide and protein degradation. We found enhanced degradation of peptide substrates in the presence of Blm10 and demonstrated that Blm10 has the capacity to accelerate proteasomal turnover of the unstructured protein tau-441 in vitro. Mechanistically, proteasome activation requires the opening of a closed gate, which allows passage of unfolded proteins into the catalytic chamber. Our data indicate that gate opening by Blm10 is achieved via engagement of its C-terminal segment with the CP. Crucial for this activity is a conserved C-terminal YYX motif, with the penultimate tyrosine playing a preeminent role. Thus, Blm10 utilizes a gate opening strategy analogous to the proteasomal ATPases HbYX-dependent mechanism. Because gating incompetent Blm10 C-terminal point mutants confers a loss of function phenotype, we propose that the cellular function of Blm10 is based on CP association and activation to promote the degradation of proteasome substrates.     

Blm10 binds to pre-activated proteasome core particles with open gate conformation

Blm10 is bound to the yeast proteasome core particle, a crucial protease of eukaryotic cells [corrected]. Two gates, at both ends of the CP, control the access of protein substrates to the catalytic cavity of the CP. Normally, substrate access is auto-inhibited by a closed gate conformation unless regulatory complexes are bound to the CP and translocate protein substrates in an ATP-dependent manner. Here, we provide evidence that Blm10 recognizes pre-activated open gate CPs, which are assumed to exist in an equilibrium with inactive closed gate CP. Consequently, single-capped Blm10-CP shows peptide hydrolysis activity. Under conditions of disturbed CP assembly, as well as in open gate mutants, pre-activated CP or constitutively active CP, respectively, prevail. Then, Blm10 sequesters disordered and open gate CP by forming double-capped Blm10(2)-CP in which peptide hydrolysis activity is repressed. We conclude that Blm10 distinguishes between gate conformations and regulates the activation of CP.     

Brh2 domain function distinguished by differential cellular responses to DNA damage and replication stress

Mutants of the fungus Ustilago maydis defective in the RecQ helicase Blm are highly sensitive to killing by the DNA replication stressor hydroxyurea. This sensitivity or toxicity is dependent on the homologous recombination (HR) system and apparently results from formation of dead-end HR DNA intermediates. HU toxicity can be suppressed by deletion of the gene encoding Brh2, the BRCA2 orthologue that serves to regulate HR by mediating Rad51 filament formation on single-stranded DNA. Brh2 harbours two different DNA-binding domains that contribute to HR function. DNA-binding activity from a single domain is sufficient to provide Brh2 functional activity in HR, but to enable HU-induced killing two functional DNA-binding domains must be present. Despite this stringent requirement for dual functioning domains, the source of DNA-binding domains is less critical in that heterologous domains can substitute for the native endogenous ones. The results suggest a model in which the nature of the DNA lesion is an important determinant in the functional response of Brh2 action.     

Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination

The breast cancer susceptibility protein, Brca2 and the RecQ helicase, Blm (Bloom syndrome mutated) are tumor suppressors that maintain genome integrity, at least in part, through homologous recombination (HR). Brca2 facilitates HR by interacting with Rad51 in multiple regions, the BRC motifs encoded by exon 11 and a single domain encoded by exon 27; however, the exact importance of these regions is not fully understood. Blm also interacts with Rad51 and appears to suppress HR in most circumstances; however, its yeast homologue Sgs1 facilitates HR in response to some genotoxins. To better understand the biological importance of these two proteins, we performed a genotoxic screen on mouse embryonic stem (ES) cells impaired for either Brca2 or Blm to establish their genotoxic profiles (a cellular dose-response to a wide range of agents). This is the first side-by-side comparison of these two proteins in an identical genetic background. We compared cells deleted for Brca2 exon 27 to cells reduced for Blm expression and find that the Brca2- and Blm-impaired cells exhibit genotoxic profiles that reflect opposing activities during HR. Cells deleted for Brca2 exon 27 are hypersensitive to γ-radiation, streptonigrin, mitomycin C and camptothecin and mildly resistant to ICRF-193 which is similar to HR defective cells null for Rad54. By contrast, Blm-impaired cells are hypersensitive to ICRF-193, mildly resistant to camptothecin and mitomycin C and more strongly resistant to hydroxyurea. These divergent profiles support the notion that Brca2 and Blm perform opposing functions during HR in mouse ES cells.