Biology subjects

Kohli, R. M.

Publications and source records attributed to Kohli, R. M..

8 recordsLinked to original sources

bioRxiv2023

The LexA-RecA* structure reveals a lock-and-key mechanism for SOS activation

The bacterial SOS response plays a key role in adaptation to DNA damage, including that caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament formed on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS response gene induction. Furthermore, we find that the interface involves three RecA monomers, with a single residue in the central monomer acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.

biochemistry↗

bioRxiv2023

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies

RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

genomics↗

bioRxiv2023

Low-input and single-cell methods for Infinium DNA methylation BeadChips

The Infinium BeadChip is the most widely used DNA methylome assay technology for population-scale epigenome profiling. However, the standard workflow requires over 200 ng of input DNA, hindering its application to small cell-number samples, such as primordial germ cells. We developed experimental and analysis workflows to extend this technology to suboptimal input DNA conditions, including ultra-low input down to single cells. DNA preamplification significantly enhanced detection rates to over 50% in five-cell samples and [~]25% in single cells. Enzymatic conversion also substantially improved data quality. Computationally, we developed a method to model the background signals influence on the DNA methylation level readings. The modified detection p-values calculation achieved higher sensitivities for low-input datasets and was validated in over 100,000 public datasets with diverse methylation profiles. We employed the optimized workflow to query the demethylation dynamics in mouse primordial germ cells available at low cell numbers. Our data revealed nuanced chromatin states, sex disparities, and the role of DNA methylation in transposable element regulation during germ cell development. Collectively, we present comprehensive experimental and computational solutions to extend this widely used methylation assay technology to applications with limited DNA.

bioinformatics↗

bioRxiv2023

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1-/-, Tet1V/V, and Tet1HxD/HxD sperm methylomes show that TET1V and TET1HxD rescue most Tet1-/- hypermethylated regions, demonstrating the importance of TET1s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

developmental biology↗

bioRxiv2022

Cooperativity between Cas9 and AID establishes broad and diversifying mutational footprints in base editors

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (1) Cas9 binding can potentially expose both DNA strands for capture by the deaminase, a feature that is enhanced by guide RNA mismatches; (2) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (3) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer; and (4) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C>T and G>A transitions over >65 bp with significant potential for targeted gene diversification.

biochemistry↗

bioRxiv2022

Engineered RecA constructs reveal the minimal SOS activation complex

The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacterias ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key SOS activation signal. Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that permit LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/510415v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@149d719org.highwire.dtl.DTLVardef@c8dd3corg.highwire.dtl.DTLVardef@1349098org.highwire.dtl.DTLVardef@fb29_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

biochemistry↗

bioRxiv2021

Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation

Oxidative modification of 5-methylcytosine (5mC) generates 5-hydroxymethylcytosine (5hmC), a DNA modification that exhibits unique epigenetic regulatory functions and impacts diverse biological processes. However, standard single-nucleus/cell bisulfite sequencing methods cannot resolve the base ambiguity between 5mC and 5hmC to accurately measure cell-type specific epigenomic patterns and gene regulatory functions of 5hmC or true 5mC. Here, we develop single-nucleus 5hmC sequencing (snhmC-seq) for quantitative and unbiased 5hmC profiling in single cells by harnessing differential deaminase activity of APOBEC3A towards 5mC and chemically protected 5hmC. We used snhmC-seq to profile single nuclei from cryopreserved mouse brain samples to reveal epigenetic heterogeneity of 5hmC at single-cell resolution and uncovered a non-canonical gene regulatory role of genic 5hmC in non-CG context.

genomics↗