Comparisons of ribonuclease HI homologs and mutants uncover a multi-state model for substrate recognition

Ribonuclease HI (RNHI) non-specifically cleaves the RNA strand in RNA:DNA hybrid duplexes in a myriad of biological processes, including retroviral reverse transcription. Several RNHI homologs contain an extended domain, termed the handle region, that is critical to substrate binding. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have suggested a kinetic model in which the handle region can exist in open (substrate-binding competent) or closed (substrate-binding incompetent) states in homologs containing arginine or lysine at position 88 (using sequence numbering of E. coli RNHI), while the handle region populates a state intermediate between the open and closed conformers in homologs with as-paragine at residue 88 [Stafford, K. A., et al., PLoS Comput. Biol. 2013, 9, 1-10]. NMR parameters characterizing handle region dynamics are highly correlated with enzymatic activity for RNHI homologs with two-state (open/closed) handle regions [Martin, J. A., et al., Biochemistry 2020, 59, 3201-3205]. The work presented herein shows that homologs with one-state (intermediate) handle regions display distinct structural features compared with their two-state counterparts. Comparisons of RNHI homologs and site-directed mutants with arginine at position 88 support a kinetic model for handle region dynamics that includes 12 unique transitions between eight conformations. Overall, these findings present an example of the structure-function relationships of enzymes and spotlight the use of NMR spectroscopy and MD simulations in uncovering fine details of conformational preferences.

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.

Kinetics of RNA and RNA:DNA hybrid strand displacement

In dynamic nucleic acids nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand which binds to a toehold, a single-stranded region on the duplex. With proper design and kinetic control, strand displacement is used to perform logic operations on molecular level to trigger the conformational change in nanostructures, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA, there has not been a comparable systematic work done for RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand) and mismatches influence the strand displacement kinetics. Through comparing the reaction rates, combined with previous theoretical studies, we observed reaction acceleration with increasing toehold length and placement of toehold at 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. Comparison of RNA displacement and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is in part explainable by the thermodynamic stabilities of the respective toehold regions, but also suggest that the rearrangement from B-form to A-form helix in case of RNA invading DNA might play a role in the kinetics. The measured kinetics of toehold-mediated strand displacement will be important in understanding and construction of more complex dynamic nucleic acid systems.

Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ

Factor-dependent transcription termination mechanisms are poorly understood. We determined a series of cryo–electron microscopy structures portraying the hexameric adenosine triphosphatase (ATPase) ρ on a pathway to terminating NusA/NusG-modified elongation complexes. An open ρ ring contacts NusA, NusG, and multiple regions of RNA polymerase, trapping and locally unwinding proximal upstream DNA. NusA wedges into the ρ ring, initially sequestering RNA. Upon deflection of distal upstream DNA over the RNA polymerase zinc-binding domain, NusA rotates underneath one capping ρ subunit, which subsequently captures RNA. After detachment of NusG and clamp opening, RNA polymerase loses its grip on the RNA:DNA hybrid and is inactivated. Our structural and functional analyses suggest that ρ, and other termination factors across life, may use analogous strategies to allosterically trap transcription complexes in a moribund state.

Structural insights into the nucleic acid remodeling mechanisms of the yeast THO-Sub2 complex

The yeast THO complex is recruited to active genes and interacts with the RNA-dependent ATPase Sub2 to facilitate the formation of mature export-competent messenger ribonucleoprotein particles and to prevent the co-transcriptional formation of RNA:DNA-hybrid-containing structures. How THO-containing complexes function at the mechanistic level is unclear. Here, we elucidated a 3.4 Å resolution structure of Saccharomyces cerevisiae THO-Sub2 by cryo-electron microscopy. THO subunits Tho2 and Hpr1 intertwine to form a platform that is bound by Mft1, Thp2, and Tex1. The resulting complex homodimerizes in an asymmetric fashion, with a Sub2 molecule attached to each protomer. The homodimerization interfaces serve as a fulcrum for a seesaw-like movement concomitant with conformational changes of the Sub2 ATPase. The overall structural architecture and topology suggest the molecular mechanisms of nucleic acid remodeling during mRNA biogenesis.

Pathogenic mutations reveal a role of RECQ4 in mitochondrial RNA:DNA hybrid formation and resolution

The synthesis of mitochondrial DNA (mtDNA) is a complex process that involves the formation and resolution of unusual nucleic acid structures, such as RNA:DNA hybrids. However, little is known about the enzymes that regulate these processes. RECQ4 is a DNA replication factor important for mtDNA maintenance, and here, we unveil a role of human RECQ4 in regulating the formation and resolution of mitochondrial RNA:DNA hybrids. Mitochondrial membrane protein p32 can block mtDNA synthesis by restricting RECQ4 mitochondrial localization via protein–protein interaction. We found that the interaction with p32 was disrupted not only by the previously reported cancer-associated RECQ4 mutation, del(A420-A463), but also by a clinical mutation of the adjacent residue, P466L. Surprisingly, although P466L mutant was present in the mitochondria at greater levels, unlike del(A420-A463) mutant, it failed to enhance mtDNA synthesis due to the accumulation of RNA:DNA hybrids throughout the mtDNA. Biochemical analysis revealed that P466L mutation enhanced RECQ4 annealing activity to generate RNA:DNA hybrids at the same time reduced its unwinding activity to resolve this structure. Hence, P466L mutation led to a reduced efficiency in completing mtDNA synthesis due to unresolved RNA:DNA hybrids across mtDNA.

XRN2 Links RNA:DNA Hybrid Resolution to Double Strand Break Repair Pathway Choice

It was recently shown that the 5’ to 3’ exoribonuclease XRN2 is involved in the DNA damage response. Importantly, loss of XRN2 abrogates DNA double stranded break repair via the non-homologous end-joining pathway. However, the mechanistic details of how XRN2 functions in the non-homologous end-joining repair process are unknown. In this study, we elucidated that XRN2-mediated RNA:DNA hybrid resolution is required to allow Ku70 binding to DNA ends. These data suggest that XRN2 is required for the initiation of non-homologous end-joining repair. Interestingly, we uncovered a role for XRN2 in the homologous recombination repair pathway. Loss of XRN2 lead to a decrease in the repair of double strand breaks by homologous recombination. Strikingly, when we removed RNA:DNA hybrids by RNaseH1 over-expression, homologous recombination was not restored. We found RNA:DNA hybrid formation at and downstream of the DSB site, suggesting that unregulated transcription inhibits homologous recombination repair. In summary, our results indicate a relation between RNA:DNA hybrid resolution and double strand break repair pathway choice.