DNA modification by sulfur: analysis of the sequence recognition specificity surrounding the modification sites - PubMed (original) (raw)

DNA modification by sulfur: analysis of the sequence recognition specificity surrounding the modification sites

Jingdan Liang et al. Nucleic Acids Res. 2007.

Abstract

The Dnd (DNA degradation) phenotype, reflecting a novel DNA modification by sulfur in Streptomyces lividans 1326, was strongly aggravated when one (dndB) of the five genes (dndABCDE) controlling it was mutated. Electrophoretic banding patterns of a plasmid (pHZ209), reflecting DNA degradation, displayed a clear change from a preferential modification site in strain 1326 to more random modifications in the mutant. Fourteen randomly modifiable sites on pHZ209 were localized, and each seemed to be able to be modified only once. Residues in a region (5'-c-cGGCCgccg-3') including a highly conserved 4-bp central core (5'-GGCC-3') in a well-documented preferential modification site were assessed for their necessity by site-directed mutagenesis. While the central core (GGCC) was found to be stringently required in 1326 and in the mutant, 'gccg' flanking its right could either abolish or reduce the modification frequency only in the mutant, and two separate nucleotides to the left had no dramatic effect. The lack of essentiality of DndB for S-modification suggests that it might only be required for enhancing or stabilizing the activity of a protein complex at the required preferential modification site, or resolving secondary structures flanking the modifiable site(s), known to constitute an obstacle for efficient modification.

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Figures

Figure 1.

Figure 1.

(A) Organization of the dnd gene cluster and in-frame deletion of _dnd_B. (B) Enhanced Dnd phenotype displayed by _dnd_B− mutant HXY2 as compared with that of wild-type 1326. Lack of Dnd phenotype displayed by _dnd_A− mutant HXY1 and _dnd_-deletion mutant HXY6 is shown as controls. (C) Preferential cleavage at specific site of EcoRV-linearized pHZ209 originating from 1326, but more random and lack of cleavage of the same DNA originating from HXY2 and HXY6, respectively. All the samples were treated with activated Tris-buffer followed by gel electrophoresis. The designation of the cleaved fragments conforms to those schematically outlined in Figure 3, with their sizes shown. DNA size markers are labeled ‘M’. Lagging of the DNA fragments behind the DNA marker could be seen as a result of overloading of the DNA samples.

Figure 2.

Figure 2.

Restoration of modification preference towards EcoRV-linearized pHZ209 (A) and partial restoration of modification preference towards EcoRV-linearized pJTU2003 (in which the nucleotide ‘C’ at position 2384 of pHZ209 was mutated to ‘A’ (B), whose DNA came from LJD1 (with integration of the _dnd_B gene) and LJD2 (with integration of the complete dnd gene cluster), in contrast to the lack of restoration originating from LJD3 (with integration of the vector pSET152 carrying no dnd gene). DNA size markers are labeled ‘M’.

Figure 3.

Figure 3.

(A) Alignment of the region traversing the modified sites (labeled as asterisks) sequenced by using pBluescript II SK(+) (left) or pMD18-T (right) as vectors for the identification of the central four nucleotides (GGCC) as the highly consensus region, which lies within one (DR2, underlining the corresponding sequence) of the three direct repeat sequences (DR1-3) in a region with a preferentially modified site (5L/R), enlarged to show the details of the region possessing three direct repeats (DR1-3, solid arrows) and two pairs of inverted repeats (IR1-2, framed arrows). Sequence tags conform to the relative positions in Figure 1C and in the linearized pHZ209 schematically presented at the bottom (B), while those labeled L or R are G site at either 2379 or 2380 in the sequenced terminus.

Figure 4.

Figure 4.

Banding patterns of pHZ2031 respectively linearized using four different but unique restriction enzymes (EcoRV, EcoRI, XhoI and NdeI) before treatment with activated Tris-buffer. Two fragments adding up to the size of linearized pHZ2031 (5.4 kb) were always seen at their expected positions after cleavage at a common modification site, as exemplified by cleavage at specific modification site 10 (Figure 3), indicated as two white asterisks in each respective gel panel. (EcoRV: 4182 and 1231; EcoRI: 3027 and 2386; XhoI: 3976 and 1437; NdeI: 4687 and 726).

Figure 5.

Figure 5.

Site-directed mutagenesis of a region traversing the consensus sequence by a two-step PCR technique involving use of two variable oligonucleotide primers (DP2 and UP3) for introducing mutations at variable positions (indicated as a star in the primer region) with two fixed primers at both ends. The 949-bp DNA with site-specific mutations (all changed to A, see text and Table 2) at variable sites (specific nucleotide numbers indicated above each gel lane) could be obtained to replace the corresponding region on pHZ209 after digestion of the respective 1187-bp PCR products with EcoRI and XhoI. The individual constructs with desired replacements (confirmed by sequencing) were introduced into 1326 (heads with no dots) and HXY2 (heads with dots) before their respective DNA samples were isolated for assay in gel A and B. M stands for markers whose sizes are shown at the left of the gel.

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