Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)n

Abstract

Submillimolar levels of calcium, similar to the physiological total (bound + free) intranuclear concentration (0.01-1 mM), induced a conformational change within d(TG/AC)n, one of the frequent dinucleotide repeats of the mammalian genome. This change is calcium-specific, because no other tested cation induced it and it was detected as a concentration-dependent transition from B- to a non-B-DNA conformation expanding from 3' end toward the 5' of the repeat. Genomic footprinting of various rat brain regions revealed the existence of similar non-B-DNA conformation within a d(TG/AC)28 repeat of the endogenous enkephalin gene only in enkephalin-expressing caudate nucleus and not in the nonexpressing thalamus. Binding assays demonstrated that DNA could bind calcium and can compete with calmodulin for calcium.

Figure 1

OsO₄ footprint of the plasmid rENKpCRII756 containing (TG)₂₈, (TC)₂₀ repeats and nonrepetitive sequences showing calcium-induced structural changes indicated by reactive thymidines. (A) Supercoiled top (lanes 2–9) and bottom (lanes 11–13) strands and linearized top strands (lanes 14–16) were probed with OsO₄ without calcium or with increasing calcium concentrations in the absence or presence of 100 mM KCl. G reaction ladders from Maxam–Gilbert sequencing of the corresponding sequences were loaded as standards (lanes 1 and 10). Solid arrowhead, 5′ reactive site at position −618; star on lane 9, Hoogsteen structure formed in the middle of (TC)₂₀ sequences at high divalent cation concentrations. (B) The positions of OsO₄ reactive thymidines in rENKpCRII756 (arrows) indicating the calcium-induced structural change, with increasing size indicating increased reactivity. The arrow pointing to the most reactive thymidine at position −618 is circled. Two reactive thymidines at the (TG)₂₈/(TC)₂₀ junction are indicated with solid circles. The star marks the central reactive thymidine of the Hoogsteen structure in the middle of (TC)₂₀; T1, T15, and T28 mark the positions of the various reactive thymidines within the (TG)₂₈ repeat. Horizontal bars mark the boundaries of the two regions within the repeat with distinct OsO₄ reactivity.

Figure 2

OsO₄ footprint of the (TG)₂₈ repeat of the rat ENK gene in the caudate nucleus and thalamus. (A) The solid arrowhead points to the most reactive thymidine at position −618 on the OsO₄ footprint performed on caudate nucleus (see also Fig. 1). The caudate nucleus (Cn) and thalamus (Th) were dissected from 40 adult rat brains and treated with OsO₄, and after the isolation of genomic DNA the 1,081-bp BglII fragment containing the endogenous (TG/AC)₂₈ repeat of the rat ENK gene (position −1,081, indicated by open arrowhead) was probed with a radioactively labeled fragment rENK505–592 (29). An identical segment of the ENK gene isolated from rat genomic DNA without OsO₄ treatment. (Lane B). Ten- and one-attomole standards (10, 1) derived from the 842-bp BglII–SphI fragment of the rENK gene (position −842 bp of the fragment is marked by arrowhead) illustrate detection sensitivity. Lanes T, C, G, and A show the sequencing ladders; the positions of the (TG)₂₈ and (TC)₂₀ repeat are marked. The sequencing primer overhangs the template segment by 8 bases; therefore, the sequencing ladder is shifted 8 bases above the genomic position. (B) Correlation between ENK mRNA levels and OsO₄ reactivity at the d(TG/AC)₂₈ repeat of the rENK gene in Cn and Th regions of the adult rat brain. Relative values were obtained from quantitive reverse transcription-coupled PCR and quantification of OsO₄ reactivity on a PhosphorImager.

Figure 3

Specificity of calcium in inducing DNA conformation change. (A) OsO₄ footprint of the supercoiled plasmid rENKpCRII (positions −505 to −756) in the presence chelators. Plasmid DNA was treated with an increasing concentration of BAPTA (lanes 3–11) that resulted in an increasingly “closed” configuration of the DNA strands at the (TG)₂₈ sequence. Chelating with EGTA (lanes 12–14) or EDTA (lanes 15–17) was effective only at higher concentrations. G indicates the Maxam–Gilbert G reaction ladder. Experimental conditions were identical to those in Fig. 1 except that chelators were added to the DNA in the indicated concentrations prior to the OsO₄ treatment. (B) The effect of other divalent cations on the DNA structure at (TG)₂₈ repeat. Calcium (lane 5), but no other divalent cation (lanes 6–8), induced DNA conformational change at the (TG)₂₈ sequence. Experimental conditions were identical to those in Fig. 1 except that the negatively supercoiled plasmid was preincubated with 700 μM of BAPTA to remove all DNA-bound calcium where indicated. Calcium, magnesium, manganese, and ferrous ion were added to the reaction mixture.

Figure 4

Calcium-binding assay. (A) Representative PhosphorImager pictures of Zeta-Probe membranes with immobilized identical slots of CaM, BSA, circular plasmid DNA [(TG)₂₈DNAcir] and linearized [(TG)₂₈DNAlin] plasmid DNA containing the (TG)₂₈ repeat, and circular plasmid DNA from which the (TG)₂₈ repeat was deleted (DNAcirc). All membranes were incubated with ⁴⁵CaCl₂ followed by the following treatments: basal binding (Basal), remaining binding after wash with 50 μM EDTA (+EDTA), remaining binding after wash with 1 mM BAPTA (+BAPTA), remaining binding after wash with 1 mM unlabeled CaCl₂ (+cold CaCl₂), binding after washing out unlabeled CaCl₂ followed by a reincubation with 50 μM ⁴⁵CaCl₂ (Reverse). (B) Graphic expression of ⁴⁵CaCl₂ binding. Data points are the mean ± SEM of relative activities of the different treatment groups normalized to nonspecific background binding (n = 3).

Figure 5

Ca²⁺/CaM-PK II autophosphorylation assay. The activity of the Ca²⁺/CaM-PK II is indicated by its phosphorylated 50- and 60-kDa subunits (I and II) in the assay. Basal activities are shown in the absence of DNA (lanes 2 and 3). Addition of 300 μM exogenous calcium increased phosphorylation (lane 4). Plasmid DNA containing residual calcium (dialyzed but no BAPTA treatment) donated calcium as indicated by increased autophosphorylation (lane 5), whereas calcium-free DNA completely inhibited autophosphorylation (lane 6). Exogenous calcium prevented the inhibitory effect of calcium-free plasmid DNA (lane 7). The specific inhibitor of CaM [CaM kinase II(290–309) CaM antagonist] completely blocked autophosphorylation (band I) (lane 8); addition of BSA (lane B) did not alter basal activity (lane 9); addition of calcium-free rat genomic DNA (lane G) also inhibited autophosphorylation (lane 10). The first band from the top in lane 9 is the result of BSA phosphorylation. Bands I and II indicate the two subunits of the enzyme; numbers indicate the molecular mass of the protein standards in kDa.