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Temozolomide (TMZ) is a methylating agent used as the first-line drug in the chemotherapy of glioblastomas. However, cancer cells eventually acquire resistance, necessitating the development of TMZ-potentiating therapy agents. TMZ induces several DNA base adducts, including O 6 -meG, 3-meA, and 7-meG. TMZ cytotoxicity stems from the ability of these adducts to directly (3-meA) or indirectly ( O 6 -meG) impair DNA replication. Although TMZ toxicity is generally attributed to O 6 -meG, other alkylated bases can be similarly important depending on the status of various DNA repair pathways of the treated cells. In this mini-review we emphasize the necessity to distinguish TMZ-sensitive glioblastomas, which do not express methylguanine-DNA methyltransferase (MGMT) and are killed by the futile cycle of mismatch repair (MMR) of the O 6 -meG/T pairs, vs. TMZ-resistant MGMT-positive or MMR-negative glioblastomas, which are selected in the course of the treatment and are killed only at higher TMZ doses by the replication-blocking 3-meA. These two types of cells can be TMZ-sensitized by inhibiting different DNA repair pathways. However, in both cases, the toxic intermediates appear to be ssDNA gaps, a vulnerability also seen in BRCA-deficient cancers. PARP inhibitors (PARPi), which were initially developed to treat BRCA1/2-deficient cancers by synthetic lethality, were re-purposed in clinical trials to potentiate the effects of TMZ. We discuss how the recent advances in our understanding of the genetic determinants of TMZ toxicity might lead to new approaches for the treatment of glioblastomas by inhibiting PARP1 and other enzymes involved in the repair of alkylation damage (e.g., APE1)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Miramova, Gartner and Ivanov.)

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug's TC-NER-dependent toxicity is needed. Here, we determine that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We map the 3'-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. Trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs are also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use of trabectedin for precision oncology and for studying TC-NER and transcription., (© 2024. The Author(s).)

Cells constantly accumulate mutations, which are caused by replication errors, as well as through the action of endogenous and exogenous DNA-damaging agents. Mutational patterns reflect the status of DNA repair machinery and the history of genotoxin exposure of a given cellular clone. Computationally derived mutational signatures can shed light on the origins of cancer. However, to understand the etiology of cancer signatures, they need to be compared with experimental signatures, which are obtained from the isogenic cell lines or organisms under controlled conditions. Experimental mutational patterns were instrumental in understanding the nature of signatures caused by mismatch repair and BRCA deficiencies. Here, we describe how different cell lines and model organisms were used in recent years to decipher mutational signatures observed in cancer genomes and provide examples of how data from different experimental systems complement and support each other., (© 2023 The Author(s).)

Mutations in the human SAMHD1 gene are known to correlate with the development of the Aicardi-Goutières syndrome (AGS), which is an inflammatory encephalopathy that exhibits neurological dysfunction characterized by increased production of type I interferon (IFN); this evidence has led to the concept that the SAMHD1 protein negatively regulates the type I IFN response. Additionally, the SAMHD1 protein has been shown to prevent efficient HIV-1 infection of macrophages, dendritic cells, and resting CD4+ T cells. To gain insights on the SAMHD1 molecular determinants that are responsible for the deregulated production of type I IFN, we explored the biochemical, cellular, and antiviral properties of human SAMHD1 mutants known to correlate with the development of AGS. Most of the studied SAMHD1 AGS mutants exhibit defects in the ability to oligomerize, decrease the levels of cellular deoxynucleotide triphosphates in human cells, localize exclusively to the nucleus, and restrict HIV-1 infection. At least half of the tested variants preserved the ability to be degraded by the lentiviral protein Vpx, and all of them interacted with RNA. Our investigations revealed that the SAMHD1 AGS variant p.G209S preserve all tested biochemical, cellular, and antiviral properties, suggesting that this residue is a determinant for the ability of SAMHD1 to negatively regulate the type I IFN response in human patients with AGS. Overall, our work genetically separated the ability of SAMHD1 to negatively regulate the type I IFN response from its ability to restrict HIV-1., (© 2017 Wiley Periodicals, Inc.)

Cohesin is a ring-shaped protein complex which comprises the Smc1, Smc3 and Scc1 subunits. It topologically embraces chromosomal DNA to connect sister chromatids and stabilize chromatin loops. It is required for proper chromosomal segregation, DNA repair and transcriptional regulation. We have recently reported that cohesin rings can adopt a "collapsed" rod-like conformation which is driven by the interaction between the Smc1 and Smc3 coiled coil arms and is regulated by post-translational modifications. The "collapsed" conformation plays a role in cohesin ring assembly and its loading on the DNA. Here we speculate about the mechanism of cohesin's conformational transitions in relation to its loading on the DNA and draw parallels with other Smc-like complexes.

SAMHD1 (sterile α motif and HD domain-containing protein 1) is a mammalian protein that regulates intracellular dNTP levels through its hydrolysis of dNTPs. SAMHD1 functions as an important retroviral restriction factor through a mechanism relying on its dNTPase activity. We and others have reported that human SAMHD1 interacts with the cell cycle regulatory proteins cyclin A, CDK1, and CDK2, which mediates phosphorylation of SAMHD1 at threonine 592, a post-translational modification that has been implicated in abrogating SAMHD1 restriction function and ability to form stable tetramers. Utilizing co-immunoprecipitation and co-localization approaches, we show that endogenous SAMHD1 is able to interact with the cyclin A-CDK1-CDK2 complexin monocytic THP-1 cells and primary monocyte-derived macrophages. Sequence analysis of SAMHD1 identifies a putative cyclin-binding motif found in many cyclin-CDK complex substrates. Using a mutagenesis-based approach, we demonstrate that the conserved residues in the putative cyclin-binding motif are important for protein expression, protein half-life, and optimal phosphorylation of SAMHD1 at Thr 592 Furthermore, we observed that SAMHD1 mutants of the cyclin-binding motif mislocalized to a nuclear compartment and had reduced ability to interact with cyclin A-CDK complexes and to form the tetramer. These findings help define the mechanisms by which SAMHD1 is phosphorylated and suggest the contribution of cyclin binding to SAMHD1 expression and stability in dividing cells., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cohesin ring, which is composed of the Smc1, Smc3, and Scc1 subunits, topologically embraces two sister chromatids from S phase until anaphase to ensure their precise segregation to the daughter cells. The opening of the ring is required for its loading on the chromosomes and unloading by the action of Wpl1 protein. Both loading and unloading are dependent on ATP hydrolysis by the Smc1 and Smc3 "head" domains, which engage to form two composite ATPase sites. Based on the available structures, we modeled the Saccharomyces cerevisiae Smc1/Smc3 head heterodimer and discovered that the Smc1/Smc3 interfaces at the two ATPase sites differ in the extent of protein contacts and stability after ATP hydrolysis. We identified smc1 and smc3 mutations that disrupt one of the interfaces and block the Wpl1-mediated release of cohesin from DNA. Thus, we provide structural insights into how the cohesin heads engage with each other., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: The approaches aimed at inhibiting the ability of cancer cells to repair DNA strand breaks have emerged as promising targets for treating cancers. Here, we assessed the potential of imipramine blue (IB), a novel analogue of antidepressant imipramine, to suppress breast cancer growth and metastasis by inhibiting the ability of breast cancer cells to repair DNA strand breaks by homologous recombination (HR)., Experimental Design: The effect of IB on breast cancer growth and metastasis was assessed in vitro as well as in preclinical mouse models. Besides, the therapeutic efficacy and safety of IB was determined in ex vivo explants from breast cancer patients. The mechanism of action of IB was evaluated by performing gene-expression, drug-protein interaction, cell-cycle, and DNA repair studies., Results: We show that the systemic delivery of IB using nanoparticle-based delivery approach suppressed breast cancer growth and metastasis without inducing toxicity in preclinical mouse models. Using ex vivo explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cells. Furthermore, our mechanistic studies revealed that IB may interact and inhibit the activity of proto-oncogene FoxM1 and associated signaling that play critical roles in HR-mediated DNA repair., Conclusions: These findings highlight the potential of IB to be applied as a safe regimen for treating breast cancer patients. Given that FoxM1 is an established therapeutic target for several cancers, the identification of a compound that inhibits FoxM1- and FoxM1-mediated DNA repair has immense translational potential for treating many aggressive cancers. Clin Cancer Res; 22(14); 3524-36. ©2016 AACR., (©2016 American Association for Cancer Research.)

Recent findings suggested that the SUMO-interacting motifs (SIMs) present in the human TRIM5α (TRIM5α(hu)) protein play an important role in the ability of TRIM5α(hu) to restrict N-MLV. Here we explored the role of SIMs in the ability of rhesus TRIM5α (TRIM5α(rh)) to restrict HIV-1, and found that TRIM5α(rh) SIM mutants IL376KK (SIM1mut) and VI405KK (SIM2mut) completely lost their ability to block HIV-1 infection. Interestingly, these mutants also lost the recently described property of TRIM5α(rh) to shuttle into the nucleus. Analysis of these variants revealed that they are unable to interact with the HIV-1 core, which might explain the reason that these variants are not active against HIV-1. Furthermore, NMR titration experiments to assay the binding between the PRYSPRY domain of TRIM5α(rh) and the small ubiquitin-like modifier 1(SUMO-1) revealed no interaction. In addition, we examined the role of SUMOylation in restriction, and find out that inhibition of SUMOylation by the adenoviral protein Gam1 did not alter the retroviral restriction ability of TRIM5α. Overall, our results do not support a role for SIMs or SUMOylation in the antiviral properties of TRIM5α., (Copyright © 2012 Elsevier Inc. All rights reserved.)

During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA., Competing Interests: The authors have declared that no competing interests exist.

Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion in vivo using degron tagging in budding yeast. The previously described DHFR-based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 "core" subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase., Competing Interests: The authors have declared that no competing interests exist.

Nuclear processes such as transcription, DNA replication and recombination are dynamically regulated by chromatin structure. Eukaryotic transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent post-translational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here we show that RAG2--an essential component of the RAG1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly--contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the mouse RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together, our results identify a new function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease.

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.

Cohesion between sister chromatids depends on a multiprotein complex called cohesin that has been proposed to hold sister DNAs together by trapping them inside a large tripartite ring. Sister chromatid cohesion has hitherto only been detected by using cytological methods in living cells. We show here that cohesion between the sister DNAs of circular minichromosomes established in vivo can be detected in vitro by velocity gradient sedimentation and agarose-gel electrophoresis. This ex vivo cohesion does not depend on intercatenation of sister DNAs but is destroyed by cleavage of cohesin's Scc1 subunit or minichromosome linearization. These data represent the best evidence so far that the cohesin ring physically holds sister DNAs together and are consistent with the notion that it does so by using a topological principle involving the trapping DNAs inside its ring.

Assembly of the HIV and other retroviruses is primarily driven by the oligomerization of the Gag polyprotein, the major viral structural protein capable of forming virus-like particles even in the absence of all other virally encoded components. Several critical determinants of Gag oligomerization are located in the C-terminal domain of the capsid protein (CA-CTD), which encompasses the most conserved segment in the highly variable Gag protein called the major homology region (MHR). The CA-CTD is thought to function as a dimerization module, although the existing model of CA-CTD dimerization does not readily explain why the conserved residues of the MHR are essential for retroviral assembly. Here we describe an x-ray structure of a distinct domain-swapped variant of the HIV-1 CA-CTD dimer stabilized by a single amino acid deletion. In the domain-swapped structure, the MHR-containing segment forms a major part of the dimerization interface, providing a structural mechanism for the enigmatic function of the MHR in HIV assembly. Our observations suggest that swapping of the MHR segments of adjacent Gag molecules may be a critical intermediate in retroviral assembly.

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 x 10(4) m(-1) s(-1) and 2.1 x 10(4) m(-1) s(-1), respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.

Sister chromatid cohesion depends on a multiprotein cohesin complex containing two SMC subunits, Smc1 and Smc3, that dimerize to form V-shaped molecules with ABC-like ATPase heads at the tips of their two arms. Cohesin's Smc1 and Smc3 "heads" are connected by an alpha kleisin subunit called Scc1, forming a tripartite ring with a diameter around 40 nm. We show here that some cohesin remains tightly bound to circular minichromosomes after their purification from yeast cells and that cleavage either of cohesin's ring or of the minichromosome's DNA destroys their association. This suggests that the stable association between cohesin and chromatin detected here is topological rather than physical, which is consistent with the notion that DNA is trapped inside cohesin rings.

The RAG1 and RAG2 proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a "core" domain that is required for the recombination reaction and a C-terminal nonessential or "non-core" region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an alpha-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.

Retroviral assembly is driven by multiple interactions mediated by the Gag polyprotein, the main structural component of the forming viral shell. Critical determinants of Gag oligomerization are contained within the C-terminal domain (CTD) of the capsid protein, which also harbors a conserved sequence motif, the major homology region (MHR), in the otherwise highly variable Gag. An unexpected clue about the MHR function in retroviral assembly emerges from the structure of the zinc finger-associated SCAN domain we describe here. The SCAN dimer adopts a fold almost identical to that of the retroviral capsid CTD but uses an entirely different dimerization interface caused by swapping the MHR-like element between the monomers. Mutations in retroviral capsid proteins and functional data suggest that a SCAN-like MHR-swapped CTD dimer forms during immature particle assembly. In the SCAN-like dimer, the MHR contributes the major part of the large intertwined dimer interface explaining its functional significance.

We present a modification of a field-cycling method which uses the NMR signal of the central transition at high field to indirectly detect zero-field quadrupole transitions. The quadrupole transitions at zero-field are detected as changes in the overall intensity of the central transition signal after the field cycle, and the method is relatively immune to lineshape distortions of the central transition caused by receiver dead time, frequency response of the probe, longer pulse lengths, etc. Cross-polarization with protons is used to enhance the central-transition signal and to increase the recycling rate of the experiment. The technique is especially useful when mixtures of several species are present. In a frozen solution of phenylboronic acid, 11B quadrupole signals of the tetrahedral species at 600 kHz and planar-trigonal species at 1450 kHz are clearly resolved. The field-cycling approach allows high-sensitivity detection of low-frequency quadrupole transitions; the experiment is sensitive enough to study boronic-acid protease inhibitors bound to proteins and may possibly be extended to lower sensitivity nuclei. The experiments are performed using a low-temperature field-cycling apparatus, operated at 10-30 K, capable of pneumatically moving the sample from the high field of a commercial 500 MHz magnet to the area above the top of the magnet where the low field is controlled by a pair of Helmholz coils.

Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

Cohesion between sister chromatids is established during S phase and maintained through G2 phase until it is resolved in anaphase (for review, see [1-3]). In Saccharomyces cerevisiae, a complex consisting of Scc1, Smc1, Smc3, and Scc3 proteins, called "cohesin," mediates the connection between sister chromatids. The evolutionary conserved yeast protein Eco1 is required for establishment of sister chromatid cohesion during S phase but not for its further maintenance during G2 or M phases or for loading the cohesin complex onto DNA. We address the molecular functions of Eco1 with sensitive sequence analytic techniques, including hidden Markov model domain fragment searches. We found a two-domain architecture with an N-terminal C2H2 Zn finger-like domain and an approximately 150 residue C-terminal domain with an apparent acetyl coenzyme A binding motif (http://mendel.imp.univie.ac.at/SEQUENCES/ECO1/). Biochemical tests confirm that Eco1 has the acetyltransferase activity in vitro. In vitro Eco1 acetylates itself and components of the cohesin complex but not histones. Thus, the establishment of cohesion between sister chromatids appears to be regulated, directly or indirectly, by a specific acetyltransferase.

Pure quadrupole resonance is a potentially useful spectroscopic approach to study the coordination of quadrupolar nuclei in biological systems. We used a field-cycling NMR method to observe boron pure quadrupole resonance of two peptide boronic acid inhibitors bound to alpha-lytic protease. The method is similar to our earlier field-cycling experiment [Ivanov, D., and Redfield, A. R. (1998) Z. Naturforsch. A 53, 269-272] but uses a simple Hartmann-Hahn transfer from proton to (11)B before field cycle and direct (11)B observe after it. Pure quadrupole resonance is sensitive to the boron coordination geometry. For example, trigonal boron in neutral phenylboronic acid, which was used as a model compound, resonates at 1450 kHz, while the resonance of the tetrahedral phenylboronic acid anion appears at approximately 600 kHz. In the complex of the MeOSuc-Ala-Ala-Pro-boroVal inhibitor with the enzyme the quadrupole resonance signal was observed at 600-650 kHz, which indicates tetrahedral boron coordination in the active site. The quadrupole frequency of the MeOSuc-Ala-Ala-Pro-boroPhe enzyme-inhibitor complex, in which a boron-histidine bond is known to be formed, was found to be the same within experimental error as in the MeOSuc-Ala-Ala-Pro-boroVal enzyme-inhibitor adduct, suggesting that the boron coordination geometry in the enzyme-MeOSuc-Ala-Ala-Pro-boroPhe adduct is also close to tetrahedral.