The PALB2 DNA-binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) tumor suppressor is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency. The PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange, a complex multistep reaction supported by only a few protein families such as RecA-like recombinases or Rad52. The mechanisms of PALB2 DNA binding and strand exchange are unknown. We performed circular dichroism, electron paramagnetic spectroscopy, and small-angle X-ray scattering analyses and determined that PALB2-DBD is intrinsically disordered, even when bound to DNA. The intrinsically disordered nature of this domain was further supported by bioinformatics analysis. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome and have many important biological functions. The complexity of the strand exchange reaction significantly expands the functional repertoire of IDPs. The results of confocal single-molecule FRET indicated that PALB2-DBD binding leads to oligomerization-dependent DNA compaction. We hypothesize that PALB2-DBD uses a chaperone-like mechanism to aid formation and resolution of complex DNA and RNA multichain intermediates during DNA replication and repair. Since PALB2-DBD alone or within the full-length PALB2 is predicted to have strong liquid-liquid phase separation (LLPS) potential, protein-nucleic acids condensates are likely to play a role in complex functionality of PALB2-DBD. Similar DNA-binding intrinsically disordered regions may represent a novel class of functional domains that evolved to function in eukaryotic nucleic acid metabolism complexes.

The PALB2 DNA-binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) tumor suppressor is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency. The PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange, a complex multistep reaction supported by only a few protein families such as RecA-like recombinases or Rad52. The mechanisms of PALB2 DNA binding and strand exchange are unknown. We performed circular dichroism, electron paramagnetic spectroscopy, and small-angle X-ray scattering analyses and determined that PALB2-DBD is intrinsically disordered, even when bound to DNA. The intrinsically disordered nature of this domain was further supported by bioinformatics analysis. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome and have many important biological functions. The complexity of the strand exchange reaction significantly expands the functional repertoire of IDPs. The results of confocal single-molecule FRET indicated that PALB2-DBD binding leads to oligomerization-dependent DNA compaction. We hypothesize that PALB2-DBD uses a chaperone-like mechanism to aid formation and resolution of complex DNA and RNA multichain intermediates during DNA replication and repair. Since PALB2-DBD alone or within the full-length PALB2 is predicted to have strong liquid-liquid phase separation (LLPS) potential, protein-nucleic acids condensates are likely to play a role in complex functionality of PALB2-DBD. Similar DNA-binding intrinsically disordered regions may represent a novel class of functional domains that evolved to function in eukaryotic nucleic acid metabolism complexes.

Acute and Long-Term Scar Characterization of Venous Ethanol Ablation in the Left Ventricular Summit.

BACKGROUND: Venous ethanol ablation (VEA) can be effective for ventricular arrhythmias from the left ventricular summit (LVS); however, there are concerns about excessive ablation by VEA. OBJECTIVES: The purpose of this study was to delineate and quantify the location, extent, and evolution of ablated tissue after VEA as an intramural ablation technique in the LVS. METHODS: VEA was performed in 59 patients with LVS ventricular arrhythmias. Targeted intramural veins were selected by electrograms from a 2F octapolar catheter or by guide-wire unipolar signals. Median ethanol delivered was 4 mL (IQR: 4-7 mL). Ablated areas were estimated intraprocedurally as increased echogenicity on intracardiac echocardiography (ICE) and incorporated into 3-dimensional maps. In 44 patients, late gadolinium enhancement cardiac magnetic resonance (CMR) imaged VEA scar and its evolution. RESULTS: ICE-demonstrated increased intramural echogenicity (median volume of 2 mL; IQR: 1.7-4.3) at the targeted region of the 3-dimensional maps. Post-ethanol CMR showed intramural scar of 2.5 mL (IQR: 2.1-3.5 mL). Early (within 48 hours after VEA) CMR showed microvascular obstruction (MVO) in 30 of 31 patients. Follow-up CMR after a median of 51 (IQR: 41-170) days showed evolution of MVO to scar. ICE echogenicity and CMR scar volumes correlated with each other and with ethanol volume. Ventricular function and interventricular septum remained intact. CONCLUSIONS: VEA leads to intramural ablation that can be tracked intraprocedurally by ICE and creates regions of MVO that are chronically replaced by myocardial scar. VEA scar volume does not compromise septal integrity or ventricular function.

Modified chordal sparing mitral valve replacement as effective technique for both stenotic and insufficient mitral valves.

BACKGROUND: Chordal apparatus preservation is important for preserving left ventricular (LV) function in the long-term perspective. We present results of originally modified chordal-sparing mitral valve replacement (MVR) successfully used in patients with mitral stenosis and mitral insufficiency. METHODS: The modified surgical method involves preserving only four strut chords with portions of the mitral valve leaflets, which are later fixed to the fibrous ring. The rest of the leaflets and marginal chords are removed. RESULTS: Starting from 1998, 484 modified universal chordal-sparing MVR were performed including 270 (55.79%) in patients with rheumatic mitral stenosis and 214 (44.21%) in patients with mitral valve insufficiency. Overall, 116 patients underwent isolated MVR, and 368 patients underwent MRV with concomitant surgical procedures. The overall in-hospital mortality was 2.5% (12 patients). Long-term efficiency was assessed in patients discharged after isolated MVR (114 patients), average follow-up period was 3.1±0.6 years. Preservation of strut chords ensured normalization of intraventricular anatomy and prevented LV dilatation; the LV Sphericity Index is maintained at 0.44-0.63. Heart failure functional class (NYHA) was improved in all patients. Non-fatal prosthesis-related complications were observed in 11 patients (9.65%). Three patients (2.63%) died due to extracardiac causes. CONCLUSIONS: The proposed modification of the strut chordal-sparing mitral valve replacement technique allows preserving functionally complete annulo-papillary apparatus, regardless of the nature of valvular dysfunction, and provides parallel movement to the mechanical prosthesis. This modified surgical technique is safe and effective and eliminates the risk of jamming of the prosthesis disk and left ventricular outflow tract obstruction.

Structural Insight into the Mechanism of PALB2 Interaction with MRG15.

The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.

Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease.

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1's ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5'-TTTTT/AA-3'. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN's sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes 'compression' near the cleavage site. This provides multiple advantages for L1-EN's role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

Analysis of RNA binding properties of human Ku protein reveals its interactions with 7SK snRNA and protein components of 7SK snRNP complex.

Human Ku heterodimeric protein composed of Ku70 and Ku80 subunits plays an important role in the non-homologous end-joining DNA repair pathway as a sensor of double strand DNA breaks. Ku is also involved in numerous cellular processes, and in some of them it acts in an RNA-dependent manner. However, RNA binding properties of the human Ku have not been well studied. Here we have analyzed interactions of a recombinant Ku heterodimer with a set of RNAs of various structure as well as eCLIP (enhanced crosslinking and immunoprecipitation) data for human Ku70. As a result, we have proposed a consensus RNA structure preferable for the Ku binding that is a hairpin possessing a bulge just near GpG sequence-containing terminal loop. 7SK snRNA is a scaffold for a ribonucleoprotein complex (7SK snRNP), which is known to participate in transcription regulation. We have shown that the recombinant Ku specifically binds a G-rich loop of hairpin 1 within 7SK snRNA. Moreover, Ku protein has been co-precipitated from HEK 293T cells with endogenous 7SK snRNA and such proteins included in 7SK snRNP as HEXIM1, Cdk9 and CTIP2. Ku and Cdk9 binding is found to be RNA-independent, meanwhile HEXIM1 and Ku co-precipitation depended on the presence of intact 7SK snRNA. The latter result has been confirmed using recombinant HEXIM1 and Ku proteins. Colocalization of Ku and CTIP2 was additionally confirmed by confocal microscopy. These results allow us to propose human Ku as a new component of the 7SK snRNP complex.

HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist.

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.

Novel RNA and DNA strand exchange activity of the PALB2 DNA binding domain and its critical role for DNA repair in cells.

BReast Cancer Associated proteins 1 and 2 (BRCA1, -2) and Partner and Localizer of BRCA2 (PALB2) protein are tumour suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. PALB2 coordinates functions of BRCA1 and BRCA2 during homology-directed repair (HDR) and interacts with several chromatin proteins. In addition to protein scaffold function, PALB2 binds DNA. The functional role of this interaction is poorly understood. We identified a major DNA-binding site of PALB2, mutations in which reduce RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the function of RAD51 recombinase. Surprisingly, it possesses the strand exchange activity without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.

RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks.

Rous sarcoma virus synaptic complex capable of concerted integration is kinetically trapped by human immunodeficiency virus integrase strand transfer inhibitors.

We determined conditions to produce milligram quantities of the soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3'-OH-recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4 °C. A C-terminal truncated IN (1-269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ∼ 151,000 Da upon Superdex 200 size-exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1-286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of the trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048, and MK-0536 are equally effective, whereas raltegravir is ∼ 70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remains associated with the trapped complex. SEC-multiangle light scattering analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this three-domain RSV IN in complex with viral DNA may be feasible.

Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding.

Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3'-to-5' directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a "closed" state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an "open" state that differs by an ∼160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.

ATP binding, ATP hydrolysis, and protein dimerization are required for RecF to catalyze an early step in the processing and recovery of replication forks disrupted by DNA damage.

In Escherichia coli, the recovery of replication following disruption by UV-induced DNA damage requires the RecF protein and occurs through a process that involves stabilization of replication fork DNA, resection of nascent DNA to allow the offending lesion to be repaired, and reestablishment of a productive replisome on the DNA. RecF forms a homodimer and contains an ATP binding cassette ATPase domain that is conserved among eukaryotic SMC (structural maintenance of chromosome) proteins, including cohesin, condensin, and Rad50. Here, we investigated the functions of RecF dimerization, ATP binding, and ATP hydrolysis in the progressive steps involved in recovering DNA synthesis following disruption by DNA damage. RecF point mutations with altered biochemical properties were constructed in the chromosome. We observed that protein dimerization, ATP binding, and ATP hydrolysis were essential for maintaining and processing the arrested replication fork, as well as for restoring DNA synthesis. In contrast, stabilization of the RecF protein dimer partially protected the DNA at the arrested fork from degradation, although overall processing and recovery remained severely impaired.

RecR-mediated modulation of RecF dimer specificity for single- and double-stranded DNA.

RecF pathway proteins play an important role in the restart of stalled replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR, and RecO initiate homologous recombination (HR) by loading of the RecA recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein. The specific role of RecF in this process is not well understood. Previous studies have proposed that RecF directs the RecOR complex to boundaries of damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA junctions. RecF belongs to ABC-type ATPases, which function through an ATP-dependent dimerization. Here, we demonstrate that the RecF of Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer, and that the DNA binding and ATPase activity of RecF depend on both the structure of DNA substrate, and the presence of RecR. We found that RecR interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to ssDNA and dimerization, likely due to increasing the ATPase rate. The DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific protein-protein interactions without significant contributions from RecR-DNA interactions. Finally, RecF neither alone nor in complex with RecR preferentially binds to the ss/dsDNA junction. Our data suggest that the specificity of the RecFOR complex toward the boundaries of DNA damaged regions may result from a network of protein-protein and DNA-protein interactions, rather than a simple recognition of the ss/dsDNA junction by RecF.

Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function.

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double-stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP-dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.

Toxicity of the staphylococcal enterotoxin B mutants with histidine-to-tyrosine substitutions.

In this study we made a series of site-directed mutants of staphylococcal enterotoxin B (SEB), in which histidine residues in the molecule were replaced by tyrosine. The mutant genes were cloned and expressed, and the corresponding proteins were purified. These mutant proteins were tested for binding to human HLA-DR4 and for mitogenetic activity in mouse splenocyte culture. Toxicity of the proteins in vivo was evaluated in the actinomycin D-primed C3H/HeJ mouse model. We found that SEB mutant proteins with fewer than four histidine-to-tyrosine (his-to-tyr) substitutions retained toxic properties similar to wild-type SEB. However, studies showed that his-to-tyr substitution of four consecutive histidine residues eliminated SEB toxicity. Our results clearly show that this genetically modified SEB protein is non-toxic and justifies its further development as a component of a new, safer vaccine to prevent SEB intoxication.

The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity.

The Escherichia coli Rep protein is a 3' to 5' SF1 DNA helicase required for replication of bacteriophage phiX174 in E. coli, and is structurally homologous to the E. coli UvrD helicase and the Bacillus stearothermophilus PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an approximately 130 degrees rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepDelta2B protein. This RepDelta2B protein can support phiX174 replication in a rep(-) E. coli strain, although the growth rate of E. coli containing the repDelta2B gene is approximately 1.5-fold slower than with the wild-type rep gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepDelta2B protein has DNA helicase activity in vitro and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity in vivo or in vitro, and that it does not facilitate DNA unwinding in vitro.

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 A resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal beta-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.