Structural basis for the recognition of sulfur in phosphorothioated DNA

Guang, Liu; Fu, Wencheng; Zhang, Zhenyi; He, Yao; Yu, Hao; Wang, Yuli; Wang, Xiaolei; Zhao, Yi‐Lei; Deng, Zixin; Wu, Geng; He, Xinyi

doi:10.1038/s41467-018-07093-1

Cited by 36 publications

(48 citation statements)

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“…ScoMcrA was named by analogy to EcoKMcrA, to highlight the shared endonuclease domains and the endonuclease activity against 5mC-modified DNA (Liu et al, 2010). However, the domain structures of EcoKMcrA and ScoMcrA are clearly different (Liu et al, 2018). EcoKMcrA consists of an N-terminal 5hmC binding domain, termed NEco, and an HNH catalytic domain.…”

Section: Sbd-sra-hnh Endonuclease Scomcramentioning

confidence: 99%

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Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

et al. 2020

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Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2-4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA. We report five SBD-HNH endonucleases, all recognizing GpsAAC/GpsTTC sequence and cleaving outside with a single nucleotide 3 stagger: EcoWI (N7/N6), Ksp11411I (N5/N4), Bsp305I (N6/N4-5), Mae9806I [N(8-10)/N(8-9)], and Sau43800I [N(8-9)/N(7-8)]. EcoWI and Bsp305I are more specific for PT modified DNA in Mg 2+ buffer, and promiscuous with Mn 2+. Ksp11411I is more PT specific with Ni 2+. EcoWI and Ksp11411I cleave fully-and hemi-PT modified oligos, while Bsp305I cleaves only fully modified ones. EcoWI forms a dimer in solution and cleaves more efficiently in the presence of two modified sites. In addition, we demonstrate that EcoWI PT-dependent activity has biological function: EcoWI expressing cells restrict dnd + GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent restriction endonucleases (PTDRs).

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Section: Sbd-sra-hnh Endonuclease Scomcramentioning

confidence: 99%

“…Sulfur binding domains (SBDs) are widespread in prokaryotic DNA binding proteins (Liu et al, 2018), but have so far not been found in eukaryotes. As the name indicates, they have a preference for sulfur in the Rp configuration in the DNA backbone.…”

Section: Introductionmentioning

confidence: 99%

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

et al. 2020

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“…This role can be inferred from the comparison with the SRA domain, which shares the PUA-like fold with EVE. The SRA domain has been characterized in considerable detail, including the base-flipping 5mC DNA-binding mechanism and characterization of its function as a modified DNA specificity module in Type IV REs which restrict DNA containing 5mC, 5hmC, and glucosylated 5hmC (Arita et al 2008;Avvakumov et al 2008;Hashimoto et al 2008;Liu et al 2018;Weigele and Raleigh 2016). Therefore, EVE domains deployed in modification-dependent restriction likely use a base-flipping mechanism, similar to that of SRA, to sense modified cytosine in various sequence contexts although some might bind derivatives of the other pyrimidine bases, thymine or uracil, which are hypermodified in some phage genomes (Weigele and Raleigh 2016).…”

Section: Eve As a Specificity Domain In Modification-dependent Restrimentioning

confidence: 99%

Modified base-binding EVE and DCD Domains Implicated in the Origins of Programmed Cell Death and the piRNA Pathway

Bell

Wolf

Koonin

2020

Preprint

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BackgroundDNA and RNA of most cellular life forms and many viruses contain an expansive repertoire of modified bases. The modified bases play diverse biological roles that include both regulation of transcription and translation, and protection against restriction endonucleases and antibiotics. Modified bases are often recognized by dedicated protein domains. However, the elaborate networks of interactions and processes mediated by modified bases are far from being completely understood.ResultsWe present a comprehensive census and classification of EVE domains that belong to the PUA/ASCH domain superfamily and bind various modified bases in DNA and RNA. Prokaryotes encode two classes of EVE domain proteins, slow-evolving and fast-evolving. The slow-evolving EVE domains in α-proteobacteria are embedded in a conserved operonic context that implies involvement in coupling between translation and respiration, in particular, cytochrome c biogenesis, potentially, via binding 5-methylcytosine in tRNAs. In β and γ-proteobacteria, the conserved associations implicate the EVE domains in the coordination of cell division, biofilm formation, and global transcriptional regulation by non-coding 6S small RNAs, which are potentially modified and bound by the EVE domains. Down-regulation of the EVE-encoding operons might cause dormancy or programmed cell death (PCD). In eukaryotes, the EVE-domain-containing THYN1-like proteins appear to inhibit PCD and regulate the cell cycle, likely, via binding 5-methylcytosine and its derivatives in DNA and/or RNA. Thus, the link between PCD and cytochrome c that appears to be universal in eukaryotes might have been inherited from the α-proteobacterial, proto-mitochondrial endosymbiont and, unexpectedly, could involve modified base recognition by EVE domains. In numerous prokaryotic genomes, fast-evolving EVE domains are embedded in defense contexts, including toxin-antitoxin modules and Type IV restriction systems, all of which can also induce PCD. These EVE domains likely recognize modified bases in invading DNA molecules and target them for restriction. We additionally identified EVE-like prokaryotic Development and Cell Death (DCD) domains that are also implicated in defense functions including PCD. This function was inherited by eukaryotes but, in animals, the DCD proteins apparently were displaced by the extended Tudor family, whose partnership with Piwi-related Argonautes became the centerpiece of the piRNA system.ConclusionsRecognition of modified bases in DNA and RNA by EVE-like domains appears to be an important, but until now, under-appreciated, common denominator in a variety of processes including PCD, cell cycle control, antivirus immunity, stress response and germline development in animals.

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“…Quantitative studies of DNA phosphorothioate modifications in prokaryotic genomes demonstrate that DNA phosphorothioate modification is widely distributed in diverse sequence contexts in bacteria and archaea [5]. The sulfur atom in R pphosphorothioated dsDNA points to the major groove of dsDNA, facilitating its identification by the sulfur-binding domain of ScoMcrA, as well as its correct cleavage, so that bacteria cannot spread and survive in other hosts [11,12]. The R p -phosphorothioated DNA functions as an antioxidant, protecting the hosting bacteria against peroxide when they live with oxidants [10].…”

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confidence: 99%

“…The sulfur atom in R pphosphorothioated dsDNA points to the major groove of dsDNA, facilitating its identification by the sulfur-binding domain of ScoMcrA, as well as its Abbreviations dnd, DNA degradation; DndE A7K , DndE variant A7K; DndE G21/24K , DndE G21/24K variant; DndE L58F and DndE L58E , DndE variants L58F and L58E; DndE WT , wild-type DndE; FP, fluorescence polarization; HTH, helix-turn-helix; K d , dissociation constants; SSRF, Shanghai Synchrotron Radiation Facility. correct cleavage, so that bacteria cannot spread and survive in other hosts [11,12].…”

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confidence: 99%

Investigating the interactions between DNA and DndE during DNA phosphorothioation

et al. 2019

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The DNA phosphorothioate modification is a novel physiological variation in bacteria. DndE controls this modification by binding to dsDNA via a mechanism that remains unclear. Structural analysis of the wild‐type DndE tetramer suggests that a positively charged region in its center is important for DNA binding. In the present study, we replaced residues G21 and G24 in this region with lysines, which increases the DNA binding affinity but does not affect the DNA degradation phenotype. Structural analysis of the mutant indicates that it forms a new tetrameric conformation and that DndE interacts with DNA as a monomer rather than as a tetramer. A structural model of the DndE–DNA complex, based on its structural homolog P22 Arc repressor, indicates that two flexible loops in DndE are determinants of DNA binding

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Structural basis for the recognition of sulfur in phosphorothioated DNA

Cited by 36 publications

References 60 publications

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

Modified base-binding EVE and DCD Domains Implicated in the Origins of Programmed Cell Death and the piRNA Pathway

Investigating the interactions between DNA and DndE during DNA phosphorothioation

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