A protein architecture guided screen for modification dependent restriction endonucleases

Abstract

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.

Figure 1.

Structure of representative PUA superfamily domains. Proteins are shown in ribbon representation in color, nucleic acids are shown in grey. (A) PUA domain of TruB (53). (B) EVE domain of Thy28 (PDB ID: 5J3E, unpublished). (C) SRA domain of UHRF1 (13). Specifically recognized modified base (5mC) is highlighted in color.

Figure 2.

Clans analysis of putative modification dependent restriction endonucleases (MDREs). Sequences of PUA superfamily domains that have been confidently assigned to families within the PUA superfamily were obtained from the Protein Data Bank (PDB), and they are designated by four letter PDB IDs. Amino acid sequences of putative modification sensing domains of enzymes in this study were added. The combined sequences were subjected to CLANS analysis (33) to visualize the extent of similarity between domains. The clusters of sequences that share the same domain family annotation were marked with shaded background.

Figure 3.

TspA15 restriction activity in vitro. (A) TspA15I digestion of phage T4GT7 (unmodified C), T4gt (5hmC) and WT T4 (g5hmC) gDNAs. Digestions were carried out in 10 mM Mg²⁺ or 1 mM Mn²⁺ buffer. (B) TspA15I digestion of C- and 5hmC-containing PCR DNA. 0.5 μg of PCR DNA (∼5.3 nM) was incubated with TspA15I enzyme in 2-fold serial dilutions, starting at 1 μg (280 nM) for 1 h in Mn²⁺ buffer. At 1/16 to 1/128 dilutions (∼17.5 nM to 2.2 nM range) the enzyme cleaves modified DNA more efficiently. The 2 log DNA ladder (2-log, 0.1 to 10 kb) was used for size marker in all presented digestions. (C) Mapping of cut/nick sites. The cut/nick sites were sequenced from 5hmC-modified PCR DNA digested by TspA15I in Mn²⁺ buffer (the base before the cut site is marked in red) and the 5hmCNG logo was generated with WebLogo (27).

Figure 4.

TspA15I phage restriction assay (phage spot test). E. coli C2566 [pTXB1-tspA15IR] cell lawns were spotted with 8 μl of the diluted phages λ_vir (10⁻² to 10⁻⁵ dilutions), T4gt and WT T4 (10⁻³ to 10⁻⁶ dilutions). Negative control: C2566 [pTXB1] vector. Positive control: C2566 [pTXB1-tagIR], expressing TagI known to restrict phage T4gt.

Figure 5.

ScoA3V digestion of pBR322 and 6mA-modified pBR322. (A) pBR322 (Dam⁺ G6mATC; Dcm⁺ C5mCWGG) and M.EcoGII (6mA) modified pBR322 were digested by ScoA3V in CutSmart buffer. The plasmids were also digested by MnlI (GAGG), DpnI (G6mATC), HpaII (CCGG), and MluCI (AATT), respectively as controls. The modified plasmid was partially resistant to MnlI and MluCI digestion. (B) Nicking sites and sequence logo derived from ScoA3V digestion of M.EcoGII-modified pBR322.

Figure 6.

Nicking activity of SRAN168-gp74 fusion endonuclease. Digestion of pBR322 (Dcm modified in C5mCWGG context), pBR322 (Dcm⁻), and pBRFM⁺ (M.Fnu4HI modified in G5mCNGC context) with the SRAN168-gp74 fusion protein, and control NEases: gp74 (phage HK97) in Mn²⁺ and Nb.BsrDI in Mg²⁺ buffer. SC, L, NC: supercoiled, linear, nicked circular DNA, respectively.

Figure 7.

Digestion of mixed PCR fragments. (A) MsiJI digestion of mixed PCR DNA substrates (unmodified DNA mixed with either 5hmC or 5mC modified DNA) in 1 mM Mn²⁺. Arrows indicate the starting DNA fragments. HpaII (CCGG) endonuclease cleaves unmodified DNA only. (B) VcaCI digestion of mixed DNA substrates (unmodified DNA mixed with 5hmC containing DNA) in 10 mM Mg²⁺.

Figure 8.

YenY4I activity assays. (A) Schematic diagram of the YenY4I domain organization (N-terminal PUA superfamily domain (PUA-sf), SauUSI-like specificity domain (DUF3427), and C-terminal PD-(D/E)XK endonuclease catalytic domain (DUF3883)). (B) YenY4I digestion of mixed PCR fragments (C+5mC, top panel; C+5hmC, bottom panel) in Mg²⁺ (buffer 2.1) or Mn²⁺ buffer (1 mM MnCl₂). Controls: HpaII digestion of regular C PCR fragment (2.9 kb); MspJI (5(h)mCNNR) digestion of 5mC or 5hmC PCR fragment (2.1 kb). YenY4I showed modified cytosine dependent activity in Mn²⁺ buffer at high enzyme concentration (2, 1, 0.5 and 0.25 μg input protein corresponding to 294, 147, 74, and 37 nM, respectively vs. 0.45 μg PCR DNA at ∼6 nM). (C) Phage restriction activity by YenY4I. Diluted λ_vir, T4gt, WT T4 phages were spotted onto the cell lawns of C2566 [pTXB1] and C2566 [pTXB1-yenY4IR]. Arrows indicate the partial restriction of phage T4gt by YenY4I. Controls: phage T4gt was strongly restricted by the expression of TspA15I or TagI. WT T4 was partially restricted by TspA15I, not restricted by TagI.

Figure 9.

Conservation scores for PUA superfamily domains mapped to the protein surface. The ConSurf server (34) in automatic mode was used to calculate conservation scores for: (A) PUA; (B) EVE and (C) SRA domains. The top panels show a complete view of the active site, the bottom panels a close-up on the putative binding site for the domain (EVE and SRA domains), or the structurally equivalent region (PUA domain).