Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

Abstract

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.

Figure 1

(A) Alignment of M.CviPII, M.CviPI, M.CviJI and M.HhaI. Motifs I through X are indicated at the bottom of the alignment and conserved residues are indicated by dots. Sequences that are identical or similar are shown in black or grey boxes, respectively. Asterisks indicate catalytic residues and hashes indicate S-adenosyl-l-methionine binding residues. (B) Alignment of Nt.CviPII and CviJI amino acid sequences. Identical residues are shown in black boxes and similar residues are shaded in grey. Two Type II restriction REase active site motifs (P-D/E-X_n-D/E/S-Z-K/E; Z = hydrophobic residue) are found in Nt.CviPII sequences: Ser126-Asp127-X₁₂-Glu139-Ile140-Lys141 and V189-Glu190-X₂₁-Glu211-Val212-Lys213. The latter motif can be partially aligned to the proposed active site of CviJI. Asterisks indicate conserved residues of the active site motif.

Figure 1

(A) Alignment of M.CviPII, M.CviPI, M.CviJI and M.HhaI. Motifs I through X are indicated at the bottom of the alignment and conserved residues are indicated by dots. Sequences that are identical or similar are shown in black or grey boxes, respectively. Asterisks indicate catalytic residues and hashes indicate S-adenosyl-l-methionine binding residues. (B) Alignment of Nt.CviPII and CviJI amino acid sequences. Identical residues are shown in black boxes and similar residues are shaded in grey. Two Type II restriction REase active site motifs (P-D/E-X_n-D/E/S-Z-K/E; Z = hydrophobic residue) are found in Nt.CviPII sequences: Ser126-Asp127-X₁₂-Glu139-Ile140-Lys141 and V189-Glu190-X₂₁-Glu211-Val212-Lys213. The latter motif can be partially aligned to the proposed active site of CviJI. Asterisks indicate conserved residues of the active site motif.

Figure 2

(A) Partial resistance to MspI challenge. pUC-cviPIIM was incubated with MspI or ScrFI. MspI does not cleave ^mCCGG whereas ScrFI cleaves ^mCCNGG. (B) Identification of methylation site. DNA sequences of PCR products derived from sodium bisulfite-treated pUC-cviPIIM were compared with those from untreated pUC-cviPIIM. The change of cytosine to thymidine corresponds to unmodified cytosine, whereas cytosine in both sodium bisulfite-treated and un-treated plasmids indicates C⁵-methylcytosine. The recognition sites stated on top of each panel are underlined in red and modified nucleotides are indicated by down arrows.

Figure 3

(A) The CviPII N-M system contains 2332 nt and two complete ORFs. The cviPIIM gene contains m5C DNA MTase motifs I through VIII as indicated (see also Figure 2). The cviPIINt gene contains two Type II REase active site motifs (see also Figure 2B). (B) Purified recombinant Nt.CviPII was analyzed on a 4–10% SDS–PAGE.

Figure 4

(A) Control of Nt.CviPII expression. Soluble extracts from equivalent induced (upper panel) and un-induced cultures (lower panel) were loaded on an SP FF column, eluted with a linear gradient of 0.1–1 M NaCl, and fractions were assayed for activity. (B) About 0.5 μg of pUC19 was incubated with 1 U of Nt.CviPII at the designated temperature for 1 h. Reactions were stopped and analyzed by electrophoresis on a 1.5% agarose gel. (C) Nt.CviPII-cleaved pUC19 and ss-M13 phage DNAs were analyzed by electrophoresis on a 6% PAG with 7 M urea. I, input; FT, flow-through; N, nicked pUC19; L, linearized pUC19; S, supercoiled pUC19; 100 bp, 100 bp DNA size marker; and LMW, low molecular weight DNA size marker.

Figure 5

Run-off sequencing of Nt.CviPII-cleaved DNA. The sequences were read as reverse-complement by the sequencing primer. Therefore, TGG corresponds to CCA, CGG to CCG, GGG to CCC and AGG to CCT. Triplet sequences in red boxes are CCN sites that are added to the substrate DNA. Those in green boxes are native CCA sequences of pUC19. Arrows under the chromatographs indicate the nt adenine added by the template-independent activity of Taq DNA polymerase used in the sequencing reactions. Arrow heads also indicate the nicking site (complement).

Figure 6

Isothermal random DNA amplification. (A) E.coli DNA was amplified using Nt.CviPII in combination with Bst DNA polymerase I large fragment (Bst), T.roseum (Tro) DNA polymerase large fragment, Vent DNA polymerase (Vent), Taq DNA polymerase (Taq) or Klenow fragment of E.coli DNA polymerase I (Klenow). (B) T.thermophilus and λ DNAs were amplified with Nt.CviPII and Bst DNA polymerase I large fragment. The amplified products were analyzed by 1.5% agarose gel electrophoresis. (C) The same cleavage reaction products as in (B) were analyzed on a 6% PAG with 7 M urea. (D) DNA was amplified from single colonies of E.coli with or without 4-base cutters CviTI (RG^CY) or MspI (C^CGG).

Figure 7

Proposed mechanism of nicking-endonuclease-mediated DNA amplification (NEMDA) using Nt.CviPII and Bst DNA polymerase I large fragment. Note: partial nicking introduced by Nt.CviPII can generate products of varying length. See Discussion for description.