A type IV modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs

Abstract

The Escherichia coli CT596 prophage exclusion genes gmrS and gmrD were found to encode a novel type IV modification-dependent restriction nuclease that targets and digests glucosylated (glc)-hydroxymethylcytosine (HMC) DNAs. The protein products GmrS (36 kDa) and GmrD (27 kDa) were purified and found to be inactive separately, but together degraded several different glc-HMC modified DNAs (T4, T2 and T6). The GMR enzyme is able to degrade both alpha-glucosy-HMC T4 DNA and beta-glucosyl-HMC T4 DNA, whereas no activity was observed against non-modified DNAs including unmodified T4 cytosine (C) DNA or non-glucosylated T4 HMC DNA. Enzyme activity requires NTP, favors UTP, is stimulated by calcium, and initially produces 4 kb DNA fragments that are further degraded to low molecular mass products. The enzyme is inhibited by the T4 phage internal protein I* (IPI*) to which it was found to bind. Overall activities of the purified GmrSD enzyme are in good agreement with the properties of the cloned gmr genes in vivo and suggest a restriction enzyme specific for sugar modified HMC DNAs. IPI* thus represents a third generation bacteriophage defense against restriction nucleases of the Gmr type.

Figure 1. Purification by affinity chromatography of GmrS and GmrD following expression of the chitin binding domain (CBD) intein self-cleaving fusion proteins

M) Protein Markers; 1) pTSC (Impact-CN gmrS expressed GmrS) Eluate I; 2) pTSC Eluate II; 3) pTDC (Impact-CN gmrD expressed GmrD) Eluate I; 4) pTDC Eluate II. The major contaminants GroEL and the uncleaved CBD/intein fusion protein are identified by Western blot, and the purified proteins are run on an SDS-PAGE stained with Coomassie blue.

Figure 2. The GmrSD enzyme digests the DNAs of phages lacking IPI*

The DNAs and internal proteins from pure phage particles were released by freeze/thawing, subjected to GmrSD digestion in the presence of 0.5 mM UTP, and run on an agarose gel stained with EthBr. M) DNA markers; 1) wtT4; 2) T2; 3) T6; 4) T4 ip1⁻ deletion mutant eG506; 5) T4 ip1⁻ point mutant (KAI⁻). The DNAs are run on an agarose gel stained with EthBr.

Figure 3. The GmrSD enzyme does not digest purified phage DNAs lacking modified cytosines and requires calcium and NTP hydrolysis for activity

Panel A: Lane 1) GmrS + T4 HMC DNA; 2) GmrSD + T4 HMC DNA; 3) GmrS + T4 C DNA; 4) GmrSD + T4 C DNA; 5) GmrS + T7 DNA; 6) GmrSD + T7 DNA; 7) GroEL Blank + T4 C DNA. Panel B: 1) GmrSD + T4 DNA (−UTP); 2) GmrS + T4DNA (−UTP); 3) GmrSD + T4 DNA (−CaCl₂); 4) GmrD + T4 DNA (−CaCl₂); 5) GmrSD + α-glc HMC DNA; 6) GmrSD + α-glc HMC DNA + GTP; 7) GmrSD + T4 DNA + UTP; 8) GmrSD + T4 DNA +GTP-γ-S; 9) GmrS + T4 DNA + GTP-γ-S. All reactions were performed at 37 °C for 30 min in 1 mM UTP, 2 mM Kacetate, 3 mM MgCl₂ and 5 mM CaCl₂ except where indicated. The DNAs are run on an agarose gel and stained with EthBr.

Figure 4. The GmrSD enzyme is able to digest α-glc, β-glc, and gentiobiosyl modified HMC containing T-even phage DNAs

M) DNA markers; 1) GroEL Blank + wtT4 DNA; 2) GmrSD + T2 DNA; 3) GmrS + T2 DNA; 4) GmrSD + T4 DNA; 5) GmrD + wtT4 DNA; 6) GmrSD + α-glcT4 DNA; 7) GmrS + α-glcT4 DNA; 8) GmrSD + β-glcT4 DNA; 9) GmrD + β-glcT4 DNA; 10) GmrSD + T6 DNA; 11) GmrS + T6 DNA; 12) GmrSD + wtT4 DNA; 13) GmrSD + wtT4 DNA(−UTP); 14) GmrS + wtT4 DNA; 15) GmrD + wtT4 DNA. All reactions were carried out at 37 °C for 30 min in 1 mM UTP, 2 mM Kacetate, 3 mM MgCl₂ and 5 mM CaCl₂ except where indicated. The DNAs are run on an agarose gel and stained with EthBr.

Figure 5. The GmrSD enzyme digests the 3 kb but not the 4.5 kb and 2.5 kb wtT4 DNA TaqI fragments

All reactions contain GmrS and GmrD and 1 mM UTP. M) DNA markers; 1) 2.5 kb fragment; 2) 3 kb fragment; 3) 4.5 kb fragment; 4) 2.5 kb + GTP-Υ-S; 5) 3.0 kb + GTP-Υ-S; 6) 4.5 kb + GTP-Y-S. All reactions were performed at 37 °C for 30 min in 1 mM UTP, 2 mM Kacetate, 3 mM MgCl₂ and 5 mM CaCl₂, or the inhibitory analogue GTP-γ-S where noted. The DNAs are run on an agarose gel and stained with EthBr.

Figure 6. The GmrS and GmrD proteins are both co-immunoprecipitated with the IPI* protein released from frozen/thawed wt T4 phage particles (wt T4 left panels) but not disrupted T4 ip1- phage lacking IPI* (8 KDa) (T4IPI⁻ right panels)

Anti-IPI* IgG is used for the immunoprecipitation. Lanes are labeled either IPI Bound (IB) for proteins immunoprecipitated or IPI Unbound (UB) for those proteins not immunoprecipitated through an association with IPI*. M) protein markers; 1) GmrS* input protein; 2) GmrD + input protein; 3) Unbound + GmrS; 4) Bound + GmrS; 5) Unbound + GmrD; 6) Bound + GmrD; 7) Unbound + GmrSD; 8) Bound + GmrSD; 9) Unbound –IPI* + GmrSD; 10) Bound –IPI* + GmrSD. The immunoprecipitates and supernatants are run on SDS-PAGE and stained with coomassie blue.

Fig. 7. Evolution of Myoviridae DNA Modifications and of GmrSD and other DNA Modification Dependent Restriction Endonucleases

Restriction endonucleases encoded by E. coli K12, Prophage P1 and numerous bacteria protect against infecting phage DNAs containing cytosine (first line). Many such enzymes are blocked by methylation or hydroxymethylation (HMC) of cytosine (line 2). The McrA and McrBC modification dependent restriction endonucleases of E. coli specifically attack HMC modified Myoviridae DNA, but are inhibited by the glucosylation of HMC (glc-HMC) (line 3). The GmrSD enzyme is able to digest the sugar modified (glc)-HMC containing DNAs of a number of T-even phages, but its activity is inhibited by the encapsidated phage IPI* protein injected with the DNA (line 4). Polymorphism of the ip1 gene family and of HMC sugar modifications among the Myoviridae suggest a widespread and incompletely characterized Gmr enzyme family.