Yong-Woon Han

ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition.

P1 ParA is a member of the Walker‐type family of partition ATPases involved in the segregation of plasmids and bacterial chromosomes. ATPases of this class interact with DNA non‐specifically in vitro and colocalize with the bacterial nucleoid to generate a variety of reported patterns in vivo. Here, we directly visualize ParA binding to DNA using total internal reflection fluorescence microscopy. This activity depends on, and is highly specific for ATP. DNA‐binding activity is not coupled to ATP hydrolysis. Rather, ParA undergoes a slow multi‐step conformational transition upon ATP binding, which licenses ParA to bind non‐specific DNA. The kinetics provide a time‐delay switch to allow slow cycling between the DNA binding and non‐binding forms of ParA. We propose that this time delay, combined with stimulation of ParAs ATPase activity by ParB bound to the plasmid DNA, generates an uneven distribution of the nucleoid‐associated ParA, and provides the motive force for plasmid segregation prior to cell division.

ParA-mediated plasmid partition driven by protein pattern self-organization

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low‐copy plasmids, such as the plasmids P1 and F, employ a three‐component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker‐type ATPase, typically called ParA, which also binds non‐specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP‐driven patterning is involved in partition is unknown. We reconstituted and visualized ParA‐mediated plasmid partition inside a DNA‐carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB‐stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion‐ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.

Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for Holliday junction branch migration

Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair. The RuvB molecular motor is an intrinsic ATP‐dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class. In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins. Based on these mutational studies and amino acid conservation among various RuvBs, we identified 10 RuvB motifs that agreed well with the features of the AAA+ protein class and that distinguished the primary structure of RuvB from that of typical DNA/RNA helicases with seven conserved helicase motifs.

Functional overlap between RecA and MgsA (RarA) in the rescue of stalled replication forks in Escherichia coli

Escherichia coli RecA protein plays a role in DNA homologous recombination, recombination repair, and the rescue of stalled or collapsed replication forks. The mgsA (rarA) gene encodes a highly conserved DNA‐dependent ATPase, whose yeast orthologue, MGS1, plays a role in maintaining genomic stability. In this study, we show a functional relationship between mgsA and recA during DNA replication. The mgsA recA double mutant grows more slowly and has lower viability than a recA single mutant, but they are equally sensitive to UV‐induced DNA damage. Mutations in mgsA and recA cause lethality in DNA polymerase I deficient cells, and suppress the temperature‐dependent growth defect of dnaE486 (Pol III α‐catalytic subunit). Moreover, recAS25P, a novel recA allele identified in this work, does not complement the slow growth of ΔmgsA ΔrecA cells or the lethality of polA12 ΔrecA, but is proficient in DNA repair, homologous recombination, SOS mutagenesis and SOS induction. These results suggest that RecA and MgsA are functionally redundant in rescuing stalled replication forks, and that the DNA repair and homologous recombination functions of RecA are separated from its function to maintain progression of replication fork.

Phage Mu Transposition Immunity: Protein Pattern Formation along DNA by a Diffusion-Ratchet Mechanism

DNA transposons integrate into host chromosomes with limited target sequence specificity. Without mechanisms to avoid insertion into themselves, transposons risk self-destruction. Phage Mu avoids this problem by transposition immunity, involving MuA-transposase and MuB ATP-dependent DNA-binding protein. MuB-bound DNA acts as an efficient transposition target, but MuA clusters bound to Mu DNA ends activate the MuB-ATPase and dissociate MuB from their neighborhood before target site commitment, making the regions near Mu ends a poor target. This MuA-cluster-MuB interaction requires formation of DNA loops between the MuA- and the MuB-bound DNA sites. At early times, MuB clusters are disassembled via loops with smaller average size, and at later times, MuA clusters find distantly located MuB clusters by forming loops with larger average sizes. We demonstrate that iterative loop formation/disruption cycles with intervening diffusional steps result in larger DNA loops, leading to preferential insertion of the transposon at sites distant from the transposon ends.

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition

Significance DNA transposons move from one genomic location to another using a transposase. A regulatory protein might assist in target selection and avoiding self-destruction. MuB is the regulatory protein of Mu transposon. Here we report that MuB is an AAA+ (ATPase associated with diverse cellular activities) ATPase and forms right-handed helical filaments around DNA. The helical parameters of MuB and DNA are mismatched and their interactions are nonuniform. We propose that enhanced ATP hydrolysis by MuB, induced by contacts with the MuA-transposon-end complex, leads to DNA deformation and bending at the MuB filament end, thus creating a favored target for transposition. MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuBs poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB’s ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein–DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.

Construction and characterization of Cy3- or Cy5-conjugated hairpin pyrrole–imidazole polyamides binding to DNA in the nucleosome

Sequence-specific DNA-binding modules, N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamides have been recently conjugated with fluorophores, and some of these conjugates could be used for the detection of specific DNA sequences. In this study, we synthesized two Py-Im polyamides 1 and 2, which interact with the 145-bp nucleosome positioning sequence 601. We conjugated the cyanine dye Cy3 or Cy5 with 1 or 2. In the absence of target DNA, the fluorescent conjugate of a Py-Im polyamide had lower fluorescence intensity compared with Cy3 or Cy5 alone. In the presence of either the target DNA or the nucleosome, the fluorescence intensity of the conjugates increased. Furthermore, we observed a Förster resonance energy transfer between the Cy3-Py-Im polyamide and the Cy5-Py-Im polyamide on the nucleosome. These results open up the possibilities that fluorescent conjugates of Py-Im polyamides can be used for characterization of the dynamic interactions within protein-DNA complexes.

Uncoupling of the ATPase activity from the branch migration activity of RuvAB protein complexes containing both wild‐type and ATPase‐defective RuvB proteins

Background:  Escherichia coli RuvAB promotes branch migration of Holliday junctions during recombination repair and homologous recombination. RuvB forms a hexameric ring through which duplex DNA passes and is translocated in an ATP‐dependent manner. ATPase‐deficient RuvB mutant K68A has a mutation in the Walker A motif and exerts a dominant‐negative effect on in vivo repair of UV‐induced DNA damage. In this study, we examined RuvAB‐dependent branch migration in the presence of a mutant RuvB, K68A.

Preferential 5‐Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein

Tet (ten-eleven translocation) family proteins oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG-methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region.

Synergistic effect of ATP for RuvA–RuvB–Holliday junction DNA complex formation

The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA–RuvB–Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA–Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA–Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA–RuvB–Holliday junction DNA complex formation.