Association of the Chromosome Replication Initiator DnaA with the Escherichia coli Inner Membrane In Vivo: Quantity and Mode of Binding

DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein’s domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication

CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR

Degradation of Intrinsically Disordered Proteins by the NADH 26S Proteasome

The 26S proteasome is the endpoint of the ubiquitin- and ATP-dependent degradation pathway. Over the years, ATP was regarded as completely essential for 26S proteasome function due to its role in ubiquitin-signaling, substrate unfolding and ensuring its structural integrity. We have previously reported that physiological concentrations of NADH are efficient in replacing ATP to maintain the integrity of an enzymatically functional 26S PC. However, the substrate specificity of the NADH-stabilized 26S proteasome complex (26S PC) was never assessed. Here, we show that the binding of NADH to the 26S PC inhibits the ATP-dependent and ubiquitin-independent degradation of the structured ODC enzyme. Moreover, the NADH-stabilized 26S PC is efficient in degrading intrinsically disordered protein (IDP) substrates that might not require ATP-dependent unfolding, such as p27, Tau, c-Fos and more. In some cases, NADH-26S proteasomes were more efficient in processing IDPs than the ATP-26S PC. These results indicate that in vitro, physiological concentrations of NADH can alter the processivity of ATP-dependent 26S PC substrates such as ODC and, more importantly, the NADH-stabilized 26S PCs promote the efficient degradation of many IDPs. Thus, ATP-independent, NADH-dependent 26S proteasome activity exemplifies a new principle of how mitochondria might directly regulate 26S proteasome substrate specificity

Recruitment of DNA Repair MRN Complex by Intrinsically Disordered Protein Domain Fused to Cas9 Improves Efficiency of CRISPR-Mediated Genome Editing

CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless, introducing directed templated changes by homology-directed repair (HDR) requires the cellular DNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process, we tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a 126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN complex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of endogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity of the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9. The new constructs improved the yield of edited cells when making endogenous point mutations or inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger insertions encoded by plasmid DNA donor templates. Improved editing was achieved with both transfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as ribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell line, exemplifying the possible implementation of our strategy in gene therapy. These constructs provide a simple approach to improve directed editing

Increase of DnaA-mCherry content in the membrane fraction as a function of its expression level.

<p><i>E. coli</i> BL21 harboring pBAD24(<i>dnaA-mCherry</i>) was induced by 0.2% arabinose for time periods shown on the graph in the inset. DnaA-mCherry content in cell fractions is presented in mCherry fluorescence intensity units, determined as described in Experimental procedures.</p

Predicted structure of <i>E. coli</i> DnaA Domains III and IV.

<p>The structure was obtained by threading the corresponding primary sequence on the structures of <i>Aquifex aeolicus</i> DnaA, DnaA Domain III of <i>Thermotoga maritima</i> and <i>E. coli</i> domain IV (Protein Data Bank accession codes 1L8Q, 2Z4R and 1J1V, respectively) using I-TASSER server (<a href="http://zhanglab.ccmb.med.umich.edu/I-TASSER/" target="_blank">http://zhanglab.ccmb.med.umich.edu/I-TASSER/</a>). In orange - Domain IIIa, containing the ATP-binding region of AAA⁺? ATPase-type proteins (Walker motif), in red - Domain IIIb, sensor region 2, in blue - linker segment that connects Domain III and IV, considered to have a membrane interaction function, and in green - the C-terminal Domain 4, DNA binding domain (for domains designation and function see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036441#pone.0036441-Kaguni1" target="_blank">[1]</a>). Amino acid residues chosen as C-terminals of different domains and used to design DnaA fragments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036441#pone-0036441-g002" target="_blank">Figure 2</a>) are shown by arrows. Images were generated using Pymol software (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p

Fluorescent structures in <i>E. coli</i> BL21 overexpressing mCherry-DnaA(117–378).

<p>Two examples (A and B) of phase-contrast (left image), fluorescence (middle) and their overlay (right) images of BL21 cells expressing mCherry-DnaA(117–378) during 4 hours. The cells visible as non-fluorescent in B actually display a fluorescence level similar to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036441#pone-0036441-g005" target="_blank">Figure 5D</a>1, that is just too low relative to intensity of inclusion bodies. Scale bar is 2 µm.</p

Combined phase-contrast and fluorescence images of <i>E. coli</i> BL21 expressing different constructs (see

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036441#pone-0036441-g002" target="_blank"><b>Figure 2</b></a><b>) of mCherry-DnaA (Left column) and inner membranes derived from corresponding cells (Right column).</b> Cells expressing mCherry alone are shown in line A. For an example of separate phase-contrast and fluorescence images see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036441#pone.0036441.s002" target="_blank">Figure S2</a>. Inner membrane vesicles are about 120 nm in size and therefore are hardly visible in homogeneously dispersed sample. However, they tend to aggregate in suspension forming large clumps visible in phase contrast. Scale bar is 2 µm.</p