Single-stranded Binding Protein Research Articles

The evolutionarily diverged single-stranded DNA-binding proteins SSB1/SSB2 differentially affect the replication, recombination, and mutation of organellar genomes in Arabidopsis thaliana

Single-stranded DNA-binding proteins (SSBs) play essential roles in the replication, recombination, and repair processes of organellar DNA molecules. In Arabidopsis thaliana, SSBs are encoded by a small family of two genes (SSB1 and SSB2). However, the functional divergence of these two SSB copies in plants remains largely unknown, and detailed studies regarding their roles in the replication and recombination of organellar genomes are still incomplete. In this study, phylogenetic, gene structure, and protein motif analyses all suggested that SSB1 and SSB2 probably diverged during the early evolution of seed plants. Based on accurate long-read sequencing results, ssb1, and ssb2 mutants had decreased copy numbers for both mitochondrial DNA (mtDNA) and plastid DNA (ptDNA), accompanied by a slight increase in structural rearrangements mediated by intermediate-sized repeats in mt genome and small-scale variants in both genomes. Our findings provide an important foundation for further investigating the effects of DNA dosage in the regulation of mutation frequencies in plant organellar genomes.

Structural and functional insights into the interaction between the bacteriophage T4 DNA processing proteins gp32 and Dda.

Bacteriophage T4 is a classic model system for studying the mechanisms of DNA processing. A key protein in T4 DNA processing is the gp32 single-stranded DNA-binding protein. gp32 has two key functions: it binds cooperatively to single-stranded DNA (ssDNA) to protect it from nucleases and remove regions of secondary structure, and it recruits proteins to initiate DNA processes including replication and repair. Dda is a T4 helicase recruited by gp32, and we purified and crystallized a gp32-Dda-ssDNA complex. The low-resolution structure revealed how the C-terminus of gp32 engages Dda. Analytical ultracentrifugation analyses were consistent with the crystal structure. An optimal Dda binding peptide from the gp32 C-terminus was identified using surface plasmon resonance. The crystal structure of the Dda-peptide complex was consistent with the corresponding interaction in the gp32-Dda-ssDNA structure. A Dda-dependent DNA unwinding assay supported the structural conclusions and confirmed that the bound gp32 sequesters the ssDNA generated by Dda. The structure of the gp32-Dda-ssDNA complex, together with the known structure of the gp32 body, reveals the entire ssDNA binding surface of gp32. gp32-Dda-ssDNA complexes in the crystal are connected by the N-terminal region of one gp32 binding to an adjacent gp32, and this provides key insights into this interaction.

The single-strand DNA-binding protein SSB1 is involved in the expression of salivary gland radiation injury repair.

Single-strand DNA-binding protein 1 (SSB1) plays a crucial role in the cellular response to DNA damage. This study aimed to explore the expression and regulation of SSB1 in normal rat salivary gland tissues and tissues following radiation, with a specific emphasis on its involvement in the repair of salivary gland injury. A total of 45 adult SD rats were randomly assigned to one control group or eight experimental groups. In the control group, five rats were euthanized without irradiation, and their parotid gland tissues were collected for analysis. The experimental groups received a dose of 6Gy of radiation targeting the head and neck region; subsequently, five rats from each group were euthanized hly to collect parotid gland tissue samples, resulting in a total of eight experimental groups. The expression levels of SSB1, γ-H2AX, and PARP1 in the parotid gland tissues were assessed via immunohistochemistry, while changes in SSB1 gene expression were quantified via RT-qPCR. No significant morphological differences were observed between the two groups following HE staining. In the immunohistochemistry (IHC) analysis, notable tissue-specific variation in SSB1 expression was identified, with higher levels detected in the ducts than in the acini and connective tissue. The expression of SSB1 gene initially increased post-radiation before subsequently decreasing, ultimately returning to baseline levels, as corroborated by the RT-qPCR results. In contrast, γ-H2AX and PARP1 exhibited minimal expression in the control group; however, their expression peaked at 1h in the experimental group before gradually declining to levels comparable to those of the control group. Radiation induces time-dependent upregulation of SSB1 expression in rat salivary glands, indicating that SSB1 may play a role in radiation-induced repair processes.

Regulation of the SOS response and homologous recombination by an integrative and conjugative element.

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer between bacteria and influence host physiology and promote evolution. ICE Bs1 of Bacillus subtilis modulates the host DNA damage response by reducing RecA filament formation. We found that the two ICE Bs1 -encoded proteins, RamT and RamA that modulate the SOS response in donors also function in recipient cells to inhibit both the SOS response and homologous recombination following transfer of the element. Expression of RamT and RamA caused a decrease in binding of the host single strand binding protein SsbA to ssDNA. We found that RamA interacted with PcrA, the host DNA translocase that functions to remove RecA from DNA, likely functioning to modulate the SOS response and recombination by stimulating PcrA activity. These findings reveal how ICE Bs1 can modulate key host processes, including the SOS response and homologous recombination, highlighting the complex interplay between mobile genetic elements and their bacterial hosts in adaptation and evolution.

Campylobacter fetus Plasmid Diversity: Comparative Analysis of Fully Sequenced Plasmids and Proposed Classification Scheme.

Campylobacter fetus is an animal pathogen that contains 2 mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius that exhibit different biochemical traits and differences in pathogenicity. Although plasmids are important in the horizontal transfer of antimicrobial resistance genes and virulence factors, C. fetus plasmids are understudied. Here, the closed sequences of 12 plasmids from Spanish C. fetus isolates were compared with the publicly available DNA sequences of C. fetus plasmids and other members of the Campylobacterales order. Sizes of C. fetus plasmids from Spanish isolates ranged between 4 and 50 kb and most of them (10/12) were potentially conjugative. Comparative analysis of the plasmids' gene content revealed a close genetic relationship between the plasmids of C. fetus isolated in Spain and those from other geographical regions, while being clearly distinct from plasmids of other Campylobacter species. Furthermore, C. fetus plasmids were grouped into two main clusters regardless of their geographic location or lineage. The distribution pattern of relaxase, replicase, and single-stranded DNA binding SSB protein encoding genes showed a clustering comparable to that resulting from plasmid whole gene content analysis, suggesting its potential use for the classification of C. fetus plasmids. Most of the larger plasmids harbored mobile genetic elements. These results can help to better understand the evolutionary dynamics and pathogenic implications of C. fetus plasmids.

Sodium Ion-Induced Structural Transition on the Surface of a DNA-Interacting Protein.

Protein surfaces have pivotal roles in interactions between proteins and other biological molecules. However, the structural dynamics of protein surfaces have rarely been explored and are poorly understood. Here, the surface of a single-stranded DNA (ssDNA) binding protein (SSB) with four DNA binding domains that bind ssDNA in binding site sizes of 35, 56, and 65 nucleotides per tetramer is investigated. Using oligonucleotides as probes to sense the charged surface, NaCl induces a two-state structural transition on the SSB surface even at moderate concentrations. Chelation of sodium ions with charged amino acids alters the network of hydrogen bonds and/or salt bridges on the surface. Such changes are associated with changes in the electrostatic potential landscape and interaction mode. These findings advance the understanding of the molecular mechanism underlying the enigmatic salt-induced transitions between different DNA binding site sizes of SSBs. This work demonstrates that monovalent salt is a key regulator of biomolecular interactions that not only play roles in non-specific electrostatic screening effects as usually assumed but also may configure the surface of proteins to contribute to the effective regulation of biomolecular recognition and other downstream events.

PRL1 interacts with and stabilizes RPA2A to regulate carbon deprivation-induced senescence in Arabidopsis.

Leaf senescence is a developmental program regulated by both endogenous and environmental cues. Abiotic stresses such as nutrient deprivation can induce premature leaf senescence, which profoundly impacts plant growth and crop yield. However, the molecular mechanisms underlying stress-induced senescence are not fully understood. In this work, employing a carbon deprivation (C-deprivation)-induced senescence assay in Arabidopsis seedlings, we identified PLEIOTROPIC REGULATORY LOCUS 1 (PRL1), a component of the NineTeen Complex, as a negative regulator of C-deprivation-induced senescence. Furthermore, we demonstrated that PRL1 directly interacts with the RPA2A subunit of the single-stranded DNA-binding Replication Protein A (RPA) complex. Consistently, the loss of RPA2A leads to premature senescence, while increased expression of RPA2A inhibits senescence. Moreover, overexpression of RPA2A reverses the accelerated senescence in prl1 mutants, and the interaction with PRL1 stabilizes RPA2A under C-deprivation. In summary, our findings reveal the involvement of the PRL1-RPA2A functional module in C-deprivation-induced plant senescence.

Single-strand DNA-binding protein suppresses illegitimate recombination in Escherichia coli, acting in synergy with RecQ helicase

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.

The RNA-binding protein PCBP1 modulates transcription by recruiting the G-quadruplex-specific helicase DHX9

PCBP1, polycytosine (poly(C)) binding protein 1, an RNA and single-stranded DNA (ssDNA) binding protein, binds poly(C) DNA tracts but it remains unclear whether its ability to bind ssDNA contributes to transcriptional regulation. Here, we report that PCBP1's DNA binding sites are enriched at transcription start sites and that by binding to promoter regions, PCBP1 regulates transcription in addition to splicing and translation. At PCBP1 target genes, we show that PCBP1 interacts with several RNA/DNA hybrid (R-loop) associated G-quadruplex resolving helicases. Furthermore, we find that PCBP1 interacts with RNA Helicase A (DHX9) to modulate transcription by regulating DHX9 accumulation and activity. PCBP1 depletion leads to defects in R-loop processing and dysregulation of transcription of PCBP1 target genes. PCBP1's high sequence specificity and interaction with helicases suggest that its mechanism in transcription involves guiding helicases to specific loci during transcription, thereby modulating their activity.

Gene editing by SSB/CRISPR-Cas9 ribonucleoprotein in bacteria

The application of CRISPR-Cas9 ribonucleoprotein (RNP) for gene editing is commonly used in plants and animals, but its application in bacteria has not been reported. In this study, we employed DNA single-strand binding protein (SSB) to construct an SSB/CRISPR-Cas9 RNP-editing system for non-homologous recombination and homologous recombination gene editing of the upp gene in bacteria. The RNP targeting the upp gene, along with SSB, was introduced into the protoplasts of Escherichia coli, Pseudomonas, and Bacillus subtilis. Transformants were obtained on plates containing 5-fluorouracil (5-FU) with gene editing efficiencies (percentage of transformants relative to the number of protoplasts) of 9.75 %, 5.02 %, and 8.37 %, respectively, and sequencing analysis confirmed 100 % non-homologous recombination. When RNP, SSB, and a 100-nucleotide single-stranded oligodeoxynucleotide (ssODN) donor were introduced into the protoplasts of these bacteria, transformants were obtained with editing efficiencies of 45.11 %, 30.13 %, and 27.18 %, respectively, and sequencing confirmed 100 % homologous recombination knockout of the upp gene. Additionally, introducing RNP, SSB, and a 100 base-pair double-stranded oligodeoxynucleotide (dsODN) donor containing a tetracycline resistance gene (tetR-dsODN) resulted in transformants on 5-FU plates with editing efficiencies of 35.94 %, 22.46 %, and 19.08 %, respectively, with sequencing confirming 100 % homologous recombination replacement of the upp gene with tetR. These results demonstrate that the SSB/CRISPR-Cas9 RNP system can efficiently, simply, and rapidly edit bacterial genomes without the need for plasmids. This study is the first to report the use of RNP-based gene editing in bacteria.

Anti-Skin Aging Potential, Antibacterial Activity, Inhibition of Single-Stranded DNA-Binding Protein, and Cytotoxic Effects of Acetone-Extracted Passiflora edulis (Tainung No. 1) Rind Extract on Oral Carcinoma Cells.

The passion fruit, Passiflora edulis, recognized for its rich nutritional properties, has long been used for its varied ethnobotanical applications. This study investigates the therapeutic potential of P. edulis var. Tainung No. 1 rind extracts by examining their polyphenolic content (TPC), total flavonoid content (TFC), anti-skin aging activities against key enzymes such as elastase, tyrosinase, and hyaluronidase, and their ability to inhibit bacterial growth, single-stranded DNA-binding protein (SSB), and their cytotoxic effects on oral carcinoma cells. The acetone extract from the rind exhibited the highest levels of TPC, TFC, anti-SSB, and antibacterial activities. The antibacterial effectiveness of the acetone-extracted rind was ranked as follows: Escherichia coli > Pseudomonas aeruginosa > Staphylococcus aureus. A titration curve for SSB inhibition showed an IC50 value of 313.2 μg/mL, indicating the potency of the acetone extract in inhibiting SSB. It also significantly reduced the activity of enzymes associated with skin aging, particularly tyrosinase, with a 54.5% inhibition at a concentration of 100 μg/mL. Gas chromatography-mass spectrometry (GC-MS) analysis tentatively identified several major bioactive compounds in the acetone extract, including stigmast-5-en-3-ol, vitamin E, palmitic acid, stigmasterol, linoleic acid, campesterol, and octadecanoic acid. Molecular docking studies suggested some of these compounds as potential inhibitors of tyrosinase and SSB. Furthermore, the extract demonstrated anticancer potential against Ca9-22 oral carcinoma cells by inhibiting cell survival, migration, and proliferation and inducing apoptosis. These results underscore the potential of P. edulis (Tainung No. 1) rind as a promising candidate for anti-skin aging, antibacterial, and anticancer applications, meriting further therapeutic investigation.

Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ.

The mitochondrial single-stranded DNA (ssDNA)binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Polγ, has been minimally explored. Here, we determined a 1.9-Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low-affinity site and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses a clinically relevant residue, R38, and a highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Polγ. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Polγ that mtSSB binds.

Cadmium causes cerebral mitochondrial dysfunction through regulating mitochondrial HSF1

Mitochondria, as the powerhouse of the cell, play a vital role in maintaining cellular energy homeostasis and are known to be a primary target of cadmium (Cd) toxicity. The improper targeting of proteins to mitochondria can compromise the normal functions of the mitochondria. However, the precise mechanism by which protein localization contributes to the development of mitochondrial dysfunction induced by Cd is still not fully understood. For this research, Hy-Line white variety chicks (1-day-old) were used and equally distributed into 4 groups: the Control group (fed with a basic diet), the Cd35 group (basic diet with 35 mg/kg CdCl2), the Cd70 group (basic diet with 70 mg/kg CdCl2) and the Cd140 group (basic diet with 140 mg/kg CdCl2), respectively for 90 days. It was found that Cd caused the accumulation of heat shock factor 1 (HSF1) in the mitochondria, and the overexpression of HSF1 in the mitochondria led to mitochondrial dysfunction and neuronal damage. This process is due to the mitochondrial HSF1 (mtHSF1), causing mitochondrial fission through the upregulation of dynamin-related protein 1 (Drp1) content, while inhibiting oligomer formation of single-stranded DNA-binding protein 1 (SSBP1), resulting in the mitochondrial DNA (mtDNA) deletion. The findings unveil an unforeseen role of HSF1 in triggering mitochondrial dysfunction.

Single-molecule characterization of SV40 replisome and novel factors: human FPC and Mcm10.

The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45-MCM-GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system.

Planarian LDB and SSDP proteins scaffold transcriptional complexes for regeneration and patterning

Sequence-specific transcription factors often function as components of large regulatory complexes. LIM-domain binding protein (LDB) and single-stranded DNA-binding protein (SSDP) function as core scaffolds of transcriptional complexes in animals and plants. Little is known about potential partners and functions for LDB/SSDP complexes in the context of tissue regeneration. In this work, we find that planarian LDB1 and SSDP2 promote tissue regeneration, with a particular function in anterior regeneration and mediolateral polarity reestablishment. We find that LDB1 and SSDP2 interact with one another and with characterized planarian LIM-HD proteins Arrowhead, Islet1, and Lhx1/5–1. We also show that SSDP2 and LDB1 function with islet1 in polarity reestablishment and with lhx1/5–1 in serotonergic neuron maturation. Finally, we find new roles for LDB1 and SSDP2 in regulating gene expression in the planarian intestine and parenchyma; these functions are likely LIM-HD-independent. Together, our work provides insight into LDB/SSDP complexes in a highly regenerative organism. Further, our work provides a strong starting point for identifying and characterizing potential binding partners of LDB1 and SSDP2 and for exploring roles for these proteins in diverse aspects of planarian physiology.

IMab antibody binds single-stranded cytosine-rich sequences and unfolds DNA i-motifs.

i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH<7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.

Single-stranded DNA binding protein hitches a ride with the Escherichia coli YoaA-χ helicase.

The Escherichia coli XPD/Rad3-like helicase, YoaA, and DNA polymerase III subunit, χ, are involved in E. coli DNA damage tolerance and repair. YoaA and χ promote tolerance to the DNA chain-terminator, 3□-azidothymidine (AZT), and together form the functional helicase complex, YoaA-χ. How YoaA-χ contributes to DNA damage tolerance is not well understood. E. coli single-stranded DNA binding protein (SSB) accumulates at stalled replication forks, and the SSB-χ interaction is required to promote AZT tolerance via an unknown mechanism. YoaA-χ and SSB interactions were investigated in vitro to better understand this DNA damage tolerance mechanism, and we discovered YoaA-χ and SSB have a functional interaction. SSB confers a substrate-specific effect on the helicase activity of YoaA-χ, barely affecting YoaA-χ on an overhang DNA substrate but inhibiting YoaA-χ on forked DNA. A paralog helicase, DinG, unwinds SSB-bound DNA in a similar manner to YoaA-χ on the substrates tested. Through use of ensemble experiments, we believe SSB binds behind YoaA-χ relative to the DNA ds/ss junction and show via single-molecule assays that SSB translocates along ssDNA with YoaA-χ. This is, to our knowledge, the first demonstration of a mechanoenzyme pulling SSB along ssDNA.

Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol β, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.

Discovery of novel disease-causing mutation in SSBP1 and its correction using adenine base editor to improve mitochondrial function

Mutations in nuclear genes regulating mitochondrial DNA (mtDNA) replication are associated with mtDNA depletion syndromes. Using whole-genome sequencing, we identified a heterozygous mutation (c.272G>A:p.Arg91Gln) in single-stranded binding protein 1 (SSBP1), a crucial protein involved in mtDNA replisome. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy. This mutation impeded multimer formation and DNA binding affinity, leading to reduced efficiency of mtDNA replication, altered mitochondria dynamics, and compromised mitochondrial function. To correct this mutation, we tested two adenine base editor (ABE) variants on patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), showed higher editing efficacy (up to 30%) and enhanced mitochondrial replication and function, despite off-target editing frequencies; however, risks from bystander editing were limited due to silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (up to 10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. Taken together, Base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.

Rice floury endosperm26 encoding a mitochondrial single-stranded DNA-binding protein is essential for RNA-splicing of mitochondrial genes and endosperm development

Endosperm, the major storage organ in cereal grains, determines the grain yield and quality. Mitochondria provide the energy for dry matter accumulation, in the endosperm development. Although mitochondrial single-stranded DNA-binding proteins (mtSSBs) play a canonical role in the maintenance of single-stranded mitochondrial DNA, their molecular functions in RNA processing and endosperm development remain obscure. Here, we report a defective rice endosperm mutant, floury endosperm26 (flo26), which develops abnormal starch grains in the endosperm. Map-based cloning and complementation experiments showed that FLO26 allele encodes a mitochondrial single-stranded DNA-binding protein, named as mtSSB1.1. Loss of function of mtSSB1.1 affects the transcriptional level of many mitochondrially-encoded genes and RNA splicing of nad1, a core component of respiratory chain complex I in mitochondria. As a result, dysfunctional mature nad1 led to dramatically decreased complex I activity, thereby reducing ATP production. Our results reveal that mtSSB1.1 plays an important role in the maintenance of mitochondrial function and endosperm development by stabilizing the splicing of mitochondrial RNA in rice.