Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

Kisiala, M.; Copelas, Alyssa; Czapinska, H.; Xu, Shuang-yong; Bochtler, Matthias

doi:10.1093/nar/gky781

Cited by 12 publications

(23 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The reason for poor methylation by the C5 MTases on ViI and phi W-14 DNA is unknown. Poor cytosine methylation may provide certain advantage against 5mC-dependent restriction systems such as Bis I, Mcr BC, Mcr A, Msp JI, and Taq I homologs (Cohen-Karni et al, 2011; Xu et al, 2016; Kisiala et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications

Flodman

Tsai

et al. 2019

Front. Microbiol.

Self Cite

View full text Add to dashboard Cite

To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5- N edU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5- N e O mdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among >200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC (TS, S=C, or G), implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine.

show abstract

Section: Resultsmentioning

confidence: 99%

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications

Flodman

Tsai

et al. 2019

Front. Microbiol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…With few exceptions, such as EcoK-McrA (7), most studies have been focused on fusion proteins featuring a domain classified as SRA, and a catalytic domain of the PD-(D/E)XK or HNH type. Examples include the SRA-PD-(D/E)XK endonucleases MspJI, AspBHI or LpnPI, the PD-(D/E)XK-SRA endonucleases PvuRts1I or AbaSI (8,9), and the SRA-HNH endonucleases ScoA3IV (Sco5333) and TagI (10,11). All these enzymes cleave DNA containing modified cytosine bases, in some cases dependent on sequence context (12).…”

Section: Introductionmentioning

confidence: 99%

“…The precise requirements for cytosine modifications differ. Some enzymes preferentially cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) (11), but not glucosyl-5-hydroxymethylcytosine (g5hmC), whereas others cleave DNA containing 5hmC and g5hmC, but not 5mC (8,9). Crystal structures show that SRA domains extrude a modified base from the DNA and scrutinize it in a dedicated pocket (13–16).…”

Section: Introductionmentioning

confidence: 99%

A protein architecture guided screen for modification dependent restriction endonucleases

Lutz

Flodman

Copelas

et al. 2019

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

show abstract

“…Thus, apo-gp74-ΔC (and likely gp74-WT) is a monomer under the conditions used for NMR resonance assignments, recording NOEs, and NMR metal-binding studies (see below). This is in contrast to some other HNH proteins, such as the putative HNH endonuclease (ORF number Gmet_0936; PDB code 4H9D) from the anaerobic Gram-negative bacterium Geobacter metallireducens (originally known as GS-15), which is a dimer at a range of concentrations (27), and the SRA-HNH endonuclease TagI and EcoKMcrA that dimerize via their HNH motifs (34,62). Because gp74 works in concert with the small and large terminase subunits (4) and the small terminase subunit is an oligomer (43), it is possible that gp74 oligomerizes in the presence of these other components of the HK97 phage DNA packaging machinery.…”

Section: Fig 1 Legend (Continued)mentioning

confidence: 88%

“…The ⍀-loop helical insert regulates binding of divalent metal ions to gp74. gp74 (5) and other HNH proteins (11,13,14,21,27,45,48,(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72) have been shown to display endonuclease activity with a variety of divalent metals. gp74 has been further shown to stimulate specific digestion of the cos site by the HK97 terminase, provided that gp74 contains an intact HNH motif (4).…”

Section: Fig 1 Legend (Continued)mentioning

confidence: 99%

HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities

et al. 2020

View full text Add to dashboard Cite

The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ββα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle. IMPORTANCE This study demonstrates that residues outside the canonical ββα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

show abstract

Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

Cited by 12 publications

References 65 publications

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications

Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications

A protein architecture guided screen for modification dependent restriction endonucleases

HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities

Contact Info

Product

Resources

About