The specificity landscape of bacterial ribonuclease P.

Developing quantitative models of substrate specificity for RNA processing enzymes is a key step toward understanding their biology and guiding applications in biotechnology and biomedicine. Optimally, models to predict relative rate constants for alternative substrates should integrate an understanding of structures of the enzyme bound to "fast" and "slow" substrates, large datasets of rate constants for alternative substrates, and transcriptomic data identifying in vivo processing sites. Such data are either available or emerging for bacterial ribonucleoprotein RNase P a widespread and essential tRNA 5' processing endonuclease, thus making it a valuable model system for investigating principles of biological specificity. Indeed, the well-established structure and kinetics of bacterial RNase P enabled the development of high throughput measurements of rate constants for tRNA variants and provided the necessary framework for quantitative specificity modeling. Several studies document the importance of conformational changes in the precursor tRNA substrate as well as the RNA and protein subunits of bacterial RNase P during binding, although the functional roles and dynamics are still being resolved. Recently, results from cryo-EM studies of E. coli RNase P with alternative precursor tRNAs are revealing prospective mechanistic relationships between conformational changes and substrate specificity. Yet, extensive uncharted territory remains, including leveraging these advances for drug discovery, achieving a complete accounting of RNase P substrates, and understanding how the cellular context contributes to RNA processing specificity in vivo.

Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P.

tRNAs are typically transcribed with extended 5' and 3' ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5' leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P.

Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid-base catalysis and substrate-dependent metal ion activation.

Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.

Kinetics of DNA strand transfer between polymerase and proofreading exonuclease active sites regulates error correction during high-fidelity replication.

We show that T7 DNA polymerase (pol) and exonuclease (exo) domains contribute to selective error correction during DNA replication by regulating bidirectional strand transfer between the two active sites. To explore the kinetic basis for selective removal of mismatches, we used a fluorescent cytosine analog (1,3-diaza-2-oxophenoxazine) to monitor the kinetics of DNA transfer between the exo and pol sites. We globally fit stopped-flow fluorescence and base excision kinetic data and compared results obtained with ssDNA versus duplex DNA to resolve how DNA transfer governs exo specificity. We performed parallel studies using hydrolysis-resistant phosphorothioate oligonucleotides to monitor DNA transfer to the exo site without hydrolysis. ssDNA binds to the exo site at the diffusion limit (109 M-1 s-1, Kd = 40 nM) followed by fast hydrolysis of the 3'-terminal nucleotide (>5000 s-1). Analysis using duplex DNA with a 3'-terminal mismatch or a buried mismatch exposed a unique intermediate state between pol and exo active sites and revealed that transfer via the intermediate to the exo site is stimulated by free nucleoside triphosphates. Transfer from the exo site back to the pol site after cleavage is fast and efficient. We propose a model to explain why buried mismatches are removed faster than single 3'-terminal mismatches and thereby provide an additional opportunity for error correction. Our data provide the first comprehensive model to explain how DNA transfer from pol to exo active sites and back again after base excision allow efficient selective mismatch removal during DNA replication to improve fidelity by more than 1000-fold.

Elucidation of the substrate of tRNA-modifying enzymes MnmEG leads to in vitro reconstitution of an evolutionarily conserved uridine hypermodification.

The evolutionarily conserved bacterial proteins MnmE and MnmG collectively install a carboxymethylaminomethyl (cmnm) group at the fifth position of wobble uridines of several tRNA species. While the reaction catalyzed by MnmEG is one of the central steps in the biosynthesis of the methylaminomethyl (mnm) posttranscriptional tRNA modification, details of the reaction remain elusive. Glycine is known to be the source of the carboxy methylamino moiety of cmnm, and a tetrahydrofolate (THF) analog is thought to supply the one carbon that is appended to the fifth position of U. However, the nature of the folate analog remains unknown. This article reports the in vitro biochemical reconstitution of the MnmEG reaction. Using isotopically labeled methyl and methylene THF analogs, we demonstrate that methylene THF is the true substrate. We also show that reduced FAD is required for the reaction and that DTT can replace the NADH in its role as a reductant. We discuss the implications of these methylene-THF and reductant requirements on the mechanism of this key tRNA modification catalyzed by MnmEG.

Intersubunit and intrasubunit interactions driving the MukBEF ATPase.

MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis-dependent DNA loop-extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity. Other structural determinants and interactions that regulate the ATPase activity of MukBEF are not clear. Here, we have investigated the MukBEF ATPase activity, identifying intersubunit and intrasubunit interactions by protein-protein crosslinking and site-specific mutagenesis. We show that interactions between the hinge of MukB and its neck region are essential for the ATPase activity, that the ParC subunit of topoisomerase IV inhibits the MukB ATPase by preventing this interaction, that MukE interaction with DNA is likely essential for viability, and that interactions between MukF and the MukB neck region are necessary for ATPase activity and viability.

Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase.

Broad evolutionary expansion of polymerase families has enabled specialization of their activities for distinct cellular roles. In addition to template-complementary synthesis, many polymerases extend their duplex products by nontemplated nucleotide addition (NTA). This activity is exploited for laboratory strategies of cloning and sequencing nucleic acids and could have important biological function, although the latter has been challenging to test without separation-of-function mutations. Several retroelement and retroviral reverse transcriptases (RTs) support NTA and also template jumping, by which the RT performs continuous complementary DNA (cDNA) synthesis using physically separate templates. Previous studies that aimed to dissect the relationship between NTA and template jumping leave open questions about structural requirements for each activity and their interdependence. Here, we characterize the structural requirements for cDNA synthesis, NTA, template jumping, and the unique terminal transferase activity of Bombyx mori R2 non-long terminal repeat retroelement RT. With sequence alignments and structure modeling to guide mutagenesis, we generated enzyme variants across motifs generally conserved or specific to RT subgroups. Enzyme variants had diverse NTA profiles not correlated with other changes in cDNA synthesis activity or template jumping. Using these enzyme variants and panels of activity assay conditions, we show that template jumping requires NTA. However, template jumping by NTA-deficient enzymes can be rescued using primer duplex with a specific length of 3' overhang. Our findings clarify the relationship between NTA and template jumping as well as additional activities of non-long terminal repeat RTs, with implications for the specialization of RT biological functions and laboratory applications.

Structural determinants and distribution of phosphate specificity in ribonucleotide reductases.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.

An autoinhibitory role for the GRF zinc finger domain of DNA glycosylase NEIL3.

The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.

Biochemical reconstitution and genetic characterization of the major oxidative damage base excision DNA repair pathway in Thermococcus kodakarensis.

Reactive oxygen species drive the oxidation of guanine to 8-oxoguanine (8oxoG), which threatens genome integrity. The repair of 8oxoG is carried out by base excision repair enzymes in Bacteria and Eukarya, however, little is known about archaeal 8oxoG repair. This study identifies a member of the Ogg-subfamily archaeal GO glycosylase (AGOG) in Thermococcus kodakarensis, an anaerobic, hyperthermophilic archaeon, and delineates its mechanism, kinetics, and substrate specificity. TkoAGOG is the major 8oxoG glycosylase in T. kodakarensis, but is non-essential. In addition to TkoAGOG, the major apurinic/apyrimidinic (AP) endonuclease (TkoEndoIV) required for archaeal base excision repair and cell viability was identified and characterized. Enzymes required for the archaeal oxidative damage base excision repair pathway were identified and the complete pathway was reconstituted. This study illustrates the conservation of oxidative damage repair across all Domains of life.

YvcI from Bacillus subtilis has in vitro RNA pyrophosphohydrolase activity.

RNA degradation is one of several ways for organisms to regulate gene expression. In bacteria, the removal of two terminal phosphate moieties as orthophosphate (Bacillus subtilis) or pyrophosphate (Escherichia coli) triggers ribonucleolytic decay of primary transcripts by 5'-monophosphate-dependent ribonucleases. In the soil-dwelling firmicute species B. subtilis, the RNA pyrophosphohydrolase BsRppH, a member of the Nudix family, triggers RNA turnover by converting primary transcripts to 5'-monophospate RNA. In addition to BsRppH, a source of redundant activity in B. subtilis has been proposed. Here, using recombinant protein expression and in vitro enzyme assays, we provide evidence for several additional RNA pyrophosphohydrolases, among them MutT, NudF, YmaB, and YvcI in B. subtilis We found that in vitro, YvcI converts RNA 5'-di- and triphosphates into monophosphates in the presence of manganese at neutral to slightly acidic pH. It preferred G-initiating RNAs and required at least one unpaired nucleotide at the 5'-end of its substrates, with the 5'-terminal nucleotide determining whether primarily ortho- or pyrophosphate is released. Exchanges of catalytically important glutamate residues in the Nudix motif impaired or abolished the enzymatic activity of YvcI. In summary, the results of our extensive in vitro biochemical characterization raise the possibility that YvcI is an additional RNA pyrophosphohydrolase in B. subtilis.

The R882H substitution in the human de novo DNA methyltransferase DNMT3A disrupts allosteric regulation by the tumor supressor p53.

A myriad of protein partners modulate the activity of the human DNA methyltransferase 3A (DNMT3A), whose interactions with these other proteins are frequently altered during oncogenesis. We show here that the tumor suppressor p53 decreases DNMT3A activity by forming a heterotetramer complex with DNMT3A. Mutational and modeling experiments suggested that p53 interacts with the same region in DNMT3A as does the structurally characterized DNMT3L. We observed that the p53-mediated repression of DNMT3A activity is blocked by amino acid substitutions within this interface, but surprisingly, also by a distal DNMT3A residue, R882H. DNMT3A R882H occurs frequently in various cancers, including acute myeloid leukemia, and our results suggest that the effects of R882H and other DNMT3A mutations may go beyond changes in DNMT3A methylation activity. To further understand the dynamics of how protein-protein interactions modulate DNMT3A activity, we determined that p53 has a greater affinity for DNMT3A than for DNMT3L and that p53 readily displaces DNMT3L from the DNMT3A:DNMT3L heterotetramer. Interestingly, this occurred even when the preformed DNMT3A:DNMT3L complex was actively methylating DNA. The frequently identified p53 substitutions (R248W and R273H), whereas able to regulate DNMT3A function when forming the DNMT3A:p53 heterotetramer, no longer displaced DNMT3L from the DNMT3A:DNMT3L heterotetramer. The results of our work highlight the complex interplay between DNMT3A, p53, and DNMT3L and how these interactions are further modulated by clinically derived mutations in each of the interacting partners.

Small-Molecule Inhibitor Screen for DNA Repair Proteins.

With the recent interest in targeting the DNA damage response (DDR) and DNA repair, new screening methodologies are needed to broaden the scope of targetable proteins beyond kinases and traditional enzymes. Many of the proteins involved in the DDR and repair impart their activity by making specific contacts with DNA. These protein-nucleic acid interactions represent a tractable target for perturbation with small molecules. We describe a high throughput, solution-based equilibrium binding fluorescence polarization assay that can be applied to a wide array of protein-nucleic acid interactions. The assay is sensitive, stable, and able to identify small molecules capable of blocking DNA-protein interactions.

The recombination mediator proteins RecFOR maintain RecA* levels for maximal DNA polymerase V Mut activity.

DNA template damage can potentially block DNA replication. Cells have therefore developed different strategies to repair template lesions. Activation of the bacterial lesion bypass DNA polymerase V (Pol V) requires both the cleavage of the UmuD subunit to UmuD' and the acquisition of a monomer of activated RecA recombinase, forming Pol V Mut. Both of these events are mediated by the generation of RecA* via the formation of a RecA-ssDNA filament during the SOS response. Formation of RecA* is itself modulated by competition with the ssDNA-binding protein (SSB) for binding to ssDNA. Previous observations have demonstrated that RecA filament formation on SSB-coated DNA can be favored in the presence of the recombination mediator proteins RecF, RecO, and RecR. We show here using purified proteins that in the presence of SSB and RecA, a stable RecA-ssDNA filament is not formed, although sufficient RecA* is generated to support some activation of Pol V. The presence of RecFOR increased RecA* generation and allowed Pol V to synthesize longer DNA products and to elongate from an unpaired primer terminus opposite template damage, also without the generation of a stable RecA-ssDNA filament.

The HIRAN domain of helicase-like transcription factor positions the DNA translocase motor to drive efficient DNA fork regression.

Helicase-like transcription factor (HLTF) is a central mediator of the DNA damage response and maintains genome stability by regressing stalled replication forks. The N-terminal HIRAN domain binds specifically to the 3'-end of single-stranded DNA (ssDNA), and disrupting this function interferes with fork regression in vitro as well as replication fork progression in cells under replication stress. Here, we investigated the mechanism by which the HIRAN-ssDNA interaction facilitates fork remodeling. Our results indicated that HIRAN capture of a denatured nascent leading 3'-end directs specific binding of HLTF to forks. DNase footprinting revealed that HLTF binds to the parental duplex ahead of the fork and at the leading edge behind the fork. Moreover, we found that the HIRAN domain is important for initiating regression of forks when both nascent strands are at the junction, but is dispensable when forks contain ssDNA regions on either template strand. We also found that HLTF catalyzes fork restoration from a partially regressed structure in a HIRAN-dependent manner. Thus, HIRAN serves as a substrate-recognition domain to properly orient the ATPase motor domain at stalled and regressed forks and initiates fork remodeling by guiding formation of a four-way junction. We discuss how these activities compare with those of two related fork remodelers, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) and zinc finger RANBP2 type-containing 3 (ZRANB3) to provide insight into their nonredundant roles in DNA damage tolerance.

The dUTPase of white spot syndrome virus assembles its active sites in a noncanonical manner.

dUTPases are essential enzymes for maintaining genome integrity and have recently been shown to play moonlighting roles when containing extra sequences. Interestingly, the trimeric dUTPase of white spot syndrome virus (wDUT) harbors a sequence insert at the position preceding the C-terminal catalytic motif V (pre-V insert), rarely seen in other dUTPases. However, whether this extra sequence endows wDUT with additional properties is unknown. Herein, we present the crystal structures of wDUT in both ligand-free and ligand-bound forms. We observed that the pre-V insert in wDUT forms an unusual ß-hairpin structure in the domain-swapping region and thereby facilitates a unique orientation of the adjacent C-terminal segment, positioning the catalytic motif V onto the active site of its own subunit instead of a third subunit. Consequently, wDUT employs two-subunit active sites, unlike the widely accepted paradigm that the active site of trimeric dUTPase is contributed by all three subunits. According to results from local structural comparisons, the active-site configuration of wDUT is similar to that of known dUTPases. However, we also found that residues in the second-shell region of the active site are reconfigured in wDUT as an adaption to its unique C-terminal orientation. We also show that deletion of the pre-V insert significantly reduces wDUT's enzymatic activity and thermal stability. We hypothesize that this rare structural arrangement confers additional functionality to wDUT. In conclusion, our study expands the structural diversity in the conserved dUTPase family and illustrates how sequence insertion and amino acid substitution drive protein evolution cooperatively.

Nonhomologous DNA end-joining for repair of DNA double-strand breaks.

Nonhomologous DNA end-joining (NHEJ) is the predominant double-strand break (DSB) repair pathway throughout the cell cycle and accounts for nearly all DSB repair outside of the S and G2 phases. NHEJ relies on Ku to thread onto DNA termini and thereby improve the affinity of the NHEJ enzymatic components consisting of polymerases (Pol µ and Pol λ), a nuclease (the Artemis·DNA-PKcs complex), and a ligase (XLF·XRCC4·Lig4 complex). Each of the enzymatic components is distinctive for its versatility in acting on diverse incompatible DNA end configurations coupled with a flexibility in loading order, resulting in many possible junctional outcomes from one DSB. DNA ends can either be directly ligated or, if the ends are incompatible, processed until a ligatable configuration is achieved that is often stabilized by up to 4 bp of terminal microhomology. Processing of DNA ends results in nucleotide loss or addition, explaining why DSBs repaired by NHEJ are rarely restored to their original DNA sequence. Thus, NHEJ is a single pathway with multiple enzymes at its disposal to repair DSBs, resulting in a diversity of repair outcomes.

Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding.

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal Sulfolobus solfataricus minichromosome maintenance (SsoMCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous Escherichia coli (Ec) DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively charged residues on the exterior surface of EcDnaB resulted in increased interaction dynamics and states compared with wild type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical for EcDnaB and SsoMCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding for EcDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.

The MukB-topoisomerase IV interaction is required for proper chromosome compaction.

The bacterial condensin MukB and the cellular decatenating enzyme topoisomerase IV interact. This interaction stimulates intramolecular reactions catalyzed by topoisomerase IV, supercoiled DNA relaxation, and DNA knotting but not intermolecular reactions such as decatenation of linked DNAs. We have demonstrated previously that MukB condenses DNA by sequestering negative supercoils and stabilizing topologically isolated loops in the DNA. We show here that the MukB-topoisomerase IV interaction stabilizes MukB on DNA, increasing the extent of DNA condensation without increasing the amount of MukB bound to the DNA. This effect does not require the catalytic activity of topoisomerase IV. Cells carrying a mukB mutant allele that encodes a protein that does not interact with topoisomerase IV exhibit severe nucleoid decompaction leading to chromosome segregation defects. These findings suggest that the MukB-topoisomerase IV complex may provide a scaffold for DNA condensation.

The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops.

MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation in vitro exhibit nucleoid decondensation in vivo.