Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding.

The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end.

Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells.

DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transposase N-terminus and the need for asymmetric transposon ends for cellular activity. We found that phosphorylation at predicted casein kinase II sites in the transposase N-terminus inhibits transposition, most likely by preventing transposase-DNA interactions. Deletion of the region containing these sites releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. When the N-terminus is deleted, the transposase gains the ability to carry out transposition using symmetric transposon left ends. This novel activity is also conferred by appending a second C-terminal domain. When combined, these modifications together result in a transposase that is highly active when symmetric transposon ends are used. Our results demonstrate that transposase N-terminal phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposition in mammalian cells. These novel insights into the mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair.

The DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1-Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker's yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1-Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1-Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

Experimental exchange of paralogous domains in the MLH family provides evidence of sub-functionalization after gene duplication.

Baker's yeast contains a large number of duplicated genes; some function redundantly, whereas others have more specialized roles. We used the MLH family of DNA mismatch repair (MMR) proteins as a model to better understand the steps that lead to gene specialization following a gene duplication event. We focused on two highly conserved yeast MLH proteins, Pms1 and Mlh3, with Pms1 having a major role in the repair of misincorporation events during DNA replication and Mlh3 acting to resolve recombination intermediates in meiosis to form crossovers. The baker's yeast Mlh3 and Pms1 proteins are significantly diverged (19% overall identity), suggesting that an extensive number of evolutionary steps, some major, others involving subtle refinements, took place to diversify the MLH proteins. Using phylogenetic and molecular approaches, we provide evidence that all three domains (N-terminal ATP binding, linker, C-terminal endonuclease/MLH interaction) in the MLH protein family are critical for conferring pathway specificity. Importantly, mlh3 alleles in the ATP binding and endonuclease domains improved MMR functions in strains lacking the Pms1 protein and did not disrupt Mlh3 meiotic functions. This ability for mlh3 alleles to complement the loss of Pms1 suggests that an ancestral Pms1/Mlh3 protein was capable of performing both MMR and crossover functions. Our strategy for analyzing MLH pathway specificity provides an approach to understand how paralogs have evolved to support distinct cellular processes.

Expanded roles for the MutL family of DNA mismatch repair proteins.

The MutL family of DNA mismatch repair proteins plays a critical role in excising and repairing misincorporation errors during DNA replication. In many eukaryotes, members of this family have evolved to modulate and resolve recombination intermediates into crossovers during meiosis. In these organisms, such functions promote the accurate segregation of chromosomes during the meiosis I division. What alterations occurred in MutL homolog (MLH) family members that enabled them to acquire these new roles? In this review, we present evidence that the yeast Mlh1-Mlh3 and Mlh1-Mlh2 complexes have evolved novel enzymatic and nonenzymatic activities and protein-protein interactions that are critical for their meiotic functions. Curiously, even with these changes, these complexes retain backup and accessory roles in DNA mismatch repair during vegetative growth.

Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex.

Intrinsically disordered regions (IDRs) are present in at least 30% of the eukaryotic proteome and are enriched in chromatin-associated proteins. Using a combination of genetics, biochemistry and single-molecule biophysics, we characterize how IDRs regulate the functions of the yeast MutLα (Mlh1-Pms1) mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablates MMR in vivo. Mlh1-Pms1 complexes with shorter IDRs that disrupt MMR retain wild-type DNA binding affinity but are impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulate the adenosine triphosphate hydrolysis and nuclease activities that are encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant MutLα in vivo. More broadly, this work highlights an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate.

The deviant ATP-binding site of the multidrug efflux pump Pdr5 plays an active role in the transport cycle.

Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites.

Mechanism of Chlorine Dioxide and Chlorate Ion Formation from the Reaction of Hypobromous Acid and Chlorite Ion.

The rate of oxidation of ClO(2)(-) by HOBr is first-order in each reactant and is general-acid-assisted in the presence of phosphate or carbonate buffers. The products are ClO(2) and ClO(3)(-), where the relative yield depends on the concentration ratio of ClO(2)(-)/OH(-). The kinetic dependence indicates the presence of a steady-state intermediate, HOBrOClO(-) (or HOBrClO(2)(-)), that undergoes general-acid-assisted reactions to generate a metastable intermediate, BrOClO (or BrClO(2)). This intermediate reacts very rapidly by two competing pathways: in one path ClO(2)(-) reacts to form 2ClO(2) and Br(-), and in the other path OH(-) (or H(2)O) reacts to form ClO(3)(-) and Br(-). Competition between these pathways determines the yield of ClO(2) but does not affect the rate of loss of HOBr. The reactions are followed by the formation of ClO(2) in the presence of excess ClO(2)(-). The rate expression for the loss of HOBr is k(1)[ClO(2)(-)][HOBr] summation operator(k(HA)[HA])/(k(-)(1) + summation operator(k(HA)[HA])), where k(1) (for the formation of the intermediate) is 97 M(-)(1) s(-)(1) and k(HA)/k(-)(1) (M(-)(1)) values, which depend on the acid (HA) strength, are 3.1 x 10(5) for H(3)O(+), 8.3 for H(2)PO(4)(-), and 0.064 for HCO(3)(-) (25.0 degrees C, &mgr; = 1.0 M). Reactions between HOBr and ClO(2)(-) are much faster than those between HOCl and ClO(2)(-).