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Results: 6

1.
Figure 1.

Figure 1. From: Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

McrBC-mediated restriction of a methyltransferase-carrying phage is alleviated by RecET-mediated homologous recombination. Lambda phage (arrow) with or without pvuIIM gene (box) on its genome (see ‘Materials and Methods’ section) was allowed to infect various E. coli strains under conditions of single infection (plaque assay). The average titer of phage without pvuIIM in the recBC sbcA mcrB::Km strain was taken as unity. Strains are: rec+ mcr+, AB1157; rec+ mcrB::Km, BNH3480; recBC sbcA mcr+, JC8679; recBC sbcA mcrB::Km, BMF1; recBC sbcA recE mcr+, BIK784; recBC mcr+, JC5519.

Ken Ishikawa, et al. Nucleic Acids Res. 2011 July;39(13):5489-5498.
2.
Figure 5.

Figure 5. From: Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

Fork cleavage requires two methylated arms. Branched DNA substrates with different methylation patterns were challenged with the McrBC enzyme at 37°C for 30 min. A reference DNA was added after the reaction was stopped to normalize, if any, uneven staining through the gel. This was followed by agarose gel electrophoresis, and the products were visualized with ethidium bromide. Ref. DNA, the reference DNA. Quantification results are provided as mean ± SD from three independent reactions.

Ken Ishikawa, et al. Nucleic Acids Res. 2011 July;39(13):5489-5498.
3.
Figure 4.

Figure 4. From: Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

Mapping cleavage sites. (A) A model replication fork (eM63++) cleaved by McrBC was subjected to polyacrylamide gel electrophoresis under denaturatingconditions (6M urea). (B) Cleavage sites. Open circle, 32P-label at 5′-end; Square, DNA methylation; Filled triangle, major product; white triangle, minor product; gray box, products corresponding to the smear. There are two labeled bands in the untreated lane, corresponding to the strand participating in the fork (293 nt), and not denaturated materials stacked in the well.

Ken Ishikawa, et al. Nucleic Acids Res. 2011 July;39(13):5489-5498.
4.
Figure 6.

Figure 6. From: Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

DNA replication fork as a target for DNA methylation and methyl-specific DNase action (a hypothesis). (A) When a methyltransferase gene is established or switched on in a cell, DNA around the replication fork is expected to be methylated earlier than elsewhere because of its higher accessibility to the methyltransferases. Although methyl-specific DNases would not cleave a replication fork with DNA methylation only in one of the two daughter duplexes near the branch point, they would cleave a fork with DNA methylation in both the arms, which represents a higher level of DNA methylation. (B) Slower replication might increase the probability of occurrence of such two-arm methylation around a fork and, therefore, the DNase-mediated cleavage. (C) Unreplicating regions might be less sensitive to the methylation. The DNase-mediated cleavage there might require higher methyltransferase activity.

Ken Ishikawa, et al. Nucleic Acids Res. 2011 July;39(13):5489-5498.
5.
Figure 2.

Figure 2. From: Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

Preparation of a long-branched DNA with methylation. The two plasmids were modified in vivo by M.FnuDII to generate 5′-m5CGCG, a McrBC recognition sequence {(i), (ii)}. Potential unmethylated plasmids were eliminated by cleavage with BstUI (5′-CGCG). pME63 was cleaved with PvuII and then with nicking endonuclease Nb.BbvCI (iii), while pMap63 was treated with nicking endonuclease Nt.BbvCI (iv). The resulting short single strands were dissociated by heating and removed by annealing with a complementary single-strand oligo DNA. The 5′-ends of intermediate (iv) were labeled with 32P (v), followed by BspHI cleavage for removal of one of the radio-labels (vii). The two DNAs with complementary single-strand regions {(vi), (vii)} were annealed to form a branched structure {viii, eM63(++)} as detailed in ‘Materials and Methods’ section. Open circle, 32P label at 5′-end; filled diamond, DNA methylation.

Ken Ishikawa, et al. Nucleic Acids Res. 2011 July;39(13):5489-5498.
6.
Figure 3.

Figure 3. From: Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

Cleavage of a model DNA replication fork by McrBC. (A) The substrate eM63++ (Figure 2A) was cleaved with McrBC, leaving products that had lost a part of the top arm, bottom arm or both. The starting material contains equal amounts of structures Precursor (vi) and Fork (viii) of Figure 2: two ethidium-stained bands in the untreated reaction (left panel, middle lane), only one of which carries 32P-label (right panel, middle lane). A portion of the material corresponds to Precursor (vii) in Figure 2; this is too faint to see by ethidium, but is visible at the bottom of the gel in the autoradiogram. This substrate was incubated with McrBC at 37°C for 30 min, followed by agarose gel electrophoresis. Precursor structure (vi) comigrates with product structure (II, top cut). Left, ethidium bromide stain; Right 32P signal. (B) Quantification of the 32P signal. Mean + SD from three independent reactions. Marker, 32P-labeled 200 bp ladder (TaKaRa).

Ken Ishikawa, et al. Nucleic Acids Res. 2011 July;39(13):5489-5498.

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