Previously, Febleomycin (BLM) has been shown to mediate RNA cleavage in a fashion more highly selective than that of DNA. Because RNAs often assume ...
Vol. 268,No.34, Issue of December 5, pp. 25909-25913, 1993 Printed in U S A A .
Tm JOUFXUOF BIOLOGICAL Cmmrs’rnu 0 1993 by T h e American Society for Biochemistry and Molecular Biology, h e .
FeeBleomycin Cleaves a Transfer RNA Precursor and Its “Transfer DNA” Analog at the Same MajorSite* (Received for publication, May 25, 1993, and in revised form, August 24, 1993)
Chris E. Holmes+ and SidneyM.Hechtg From the Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia22901
much greater selectivity than DNA strand scission, the two Previously,Febleomycin (BLM) hasbeenshownto mediate RNA cleavage in a fashion more highly selective processes also have key features in common. In particular, than that of DNA. Because RNAs often assume second- BLM-mediated RNA degradation was also shown to be oxidaary and tertiary structures not commonly encounteredtive in nature and to afford strand scission products some of with DNAs, it was not clear whether the greater selec- which can also be envisioned to form via abstraction of the tivity of RNA cleavage was a consequence of differences ribose C-4’ H by activated FeSBLM (10, 14). Relative to DNA, in the mononucleotide constituents RNA of and DNA,or RNA has at least three potential advantages asa therapeutic of the three-dimensional structures of the individual target for BLM. These include the greater selectivity of RNA substrates. Accordingly, we prepared a ‘%DNA”identical cleavage, the greater accessibility of (cytoplasmic) RNA to exin sequence with Bacillus subtilie tRNAH’*precursor, ogenous agents, and the dearth of cellular mechanismsfor RNA the latter of which is known to be a good substrate for repair. Fe(I1)-BLMAz and which undergoes oxidative cleavage Although the greater selectivity of RNA cleavage by BLM predominantly atUse. Remarkably, the tDNA underwent might logically be ascribed to intrinsic differences in the susP.At higher concentrations cleavage predominantly at of Fe(II)-BLMA*, the tDNA was extensively degraded, ceptibility of DNA and RNA to BLM-mediated destruction, it while the tRNAHis precursor was not. Competition ex- may be noted that the RNA substrates studied thus far are believed to havesecondary and tertiary structures unlike those periments suggested that this was not due to more efficient binding of FeBLM to the tDNA; in fact the tRNA found in B-form DNA, the “usual”DNA substrate for BLM. In fact, it has been noted that structural alteration of DNA via precursor appeared to be bound more efficiently. The lesser cleavageof the tRNAHis may be due to limitations methylation (16-181, platination (19,201, or the introductionof bulges in theduplex (21) substantially alters theobserved patin thefacility ofchemicaltransformationfollowing FeBLM binding, orelse to the formation RNA of lesions tern of BLM-mediated degradation. that do not lead directly to RNA strand scission. To facilitate a comparison of RNA and DNA cleavage by BLM, we have prepared a single-stranded DNA molecule having the same primary structureas Bacillus subtilistRNAHiB precursor The bleomycins are clinically useful antitumor agents having(Fig. 11, the latter of which is a particularly good substrate for unique chemical and biochemical properties (1-3). The latter cleavage by Fe(II).BLM and is cleaved predominantly at a have been studied intensively in an effort to define the bio- single site (10-12). Presently, we demonstrate that the tRNA chemical loci at which bleomycin mediates its therapeutic ef- and ”tDNA” substrates are cleaved at the same major site by fects, and to characterizeits behavior at those loci. Fe.BLM, an observation with important implications for the Although a few different therapeutic targets were suggestednature of substrate recognition by BLM. for BLM’ in early studies, most efforts have focused on the EXPERIMENTALPROCEDURES ability of the drug to mediate the oxidative destruction of DNA (2, 3). This process, whichinvolves a n “activated” species Materials formed from BLM, a redox-active metal ion such as iron or Blenoxane, obtained from Bristol Laboratories, was fractionated as copper, and 02,produces lesions in chromosomal DNA that described t o afford bleomycin Az (22). A pSP64 plasmid encoding B . include single- and double-stranded breaks as well as alkalisubtilis tRNAHi’ precursor was kindly provided by Dr. Barbara Vold labile lesions (2-4). These structural alterations,all or most of (SRI International, Menlo Park, CA). Calf intestinal phosphatase and which can be repaired ( 5 ) , are believed to form predominantly restriction endonuclease EcoRI were purchased from Boehringer Mannat a subset of the 5‘-GC-3‘ and 5‘-GT-3‘ sequences in DNA heim;T4 DNA ligase and T4polynucleotide kinase werefrom Life Technologies Inc. SP6 RNA polymerase was purchased from Promega; (6-8). Recently, it has been shownconvincingly that BLM can also [y-32PlATPwasobtainedfromICNRadiochemicals.Theprotected effect RNA strand scission (9-15). Althoughoccurring with nucleotides for tDNA synthesis were from Milligen Biosearch.
* This work was supported in part by United States Public Health Service Research Grant CA53913,awardedby the National Cancer Institute. The costsof publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“advertisement” in accordancewith18U.S.C.Section1734 solely to indicate this fact. $ Pratt Fellowship, 1988-1989; Governor’s Fellowship, 1990-1991; DuPont Graduate Fellowship, 1990-1991. 0 To whom correspondence should be addressed: Dept.of Chemistry, University of Virginia, Charlottesville,VA 22901. The abbreviationsused are: BLM, bleomycin; tDNAHi8 precursor, a DNA molecule havingthe same primary sequence B. assubtilis tRNAHi* precursor; nt, nucleotide.
Methods Preparation of 5’-32P-EndLabeled tRNAHia Precursor-The plasmid encodingtRNAHiaprecursor (23) wasisolatedfromEscherichiacoli JMlOl as described (24).The tRNAHi” precursor was prepared from the EcoRI-linearized DNA plasmid using SP6 RNA polymerase (25). The RNA transcripts were isolated, dephosphorylated using calfintestinal phosphatase, and 5‘-end labeled in the presence of T4 polynucleotide kinase and [Y-~~PIATP, essentially as described (14). Preparation of 5’-32P-EndLabeled tDNA Precursor Substrate-The tDNA substrate was preparedby the enzymatic manipulationof chemically synthesizedDNA fragments. Initiallythree DNA oligonucleotides were prepared on a Biosearch 7800 series DNA synthesizer usingstandard phosphoramidate chemistry, and then purified on a NENSORB
25909
Cleavage of tRNAHisPrecursor and tDNAHisby Fe-Bleomycin
25910
AAAUAAAAAUUGAAUU" C C "GAAUACAAGCUVUAUCXAUAUG&JUU&0CG-
C-G A-U 0°C A- U
u
C
AAATAAAAATTQAATT" C
5
'
~
~
~ C
C G C
~
~
~
~
~
' Q - C c "rC-0 0-C
C-Q
A-T Q- C *A- 1 T C T O
c
U G G UG
Q
p
1. Structures ofB. subtilie tRNAmmprecursor anda tDNA arbitrarily folded in the same secondary structure. In addition to the major sites of cleavage at US6( P ) , denoted by large arrows, other significant sites of tDNA cleavageare indicated by small arrows. Minor sites of cleavage of both substrates are denoted by asterisks. FIG.
preparative cartridge, essentially as described (26). These three oligonucleotides had the sequences shown below: 62-nt fragment-
5'-GAATACAAGC"ATCAATATATATGCmGGCGG-
62nt
56nt
8 3 " 5
+
+
3'
TI'GTGGCGAAGTGG'ITAACGCACCAGATTGT-3' 56-nt fragment27-nt fragment-
GCGT-3' Both the 62- and 56-nt fragments, corresponding to the 5'- and 3'"halves" of the tDNA substrate, respectively, were separately 5'-phosphorylated using T4 polynucleotide kinase and either 1m~ unlabeled ATP (56-nt fragment) or 0.5 mCi of [ysZPIATP(62-nt fragment). The three DNA oligonucleotides were heated a t 85 "C for 1 min in 50 m~ NaCl then cooled to room temperature over a period of2-3 h to effect annealing of the three strands.Ligation of the 62- and 56-ntfragments was accomplishedin a reaction mixture (100 pl total volume)containing 50 m~ Tris-HC1, pH 7.6, 30 m~ NaCl, 1.4 m~ ATP, 5 m~ 2-mercaptoethanol, 2 m~ dithiothreitol, 10 m~ MgClZ, polyethyleneglycol(2% w/v, PEG-8000)and 12 units of T4 DNAligase (1unit catalyzes the exchange of 1 nmol of 82P-labeledpyrophosphate into ATP in 20 min at 37 "C). The reaction mixture was maintained at 0 "C for 2 h, then a t room temperature for 12 h. The tDNA product was purified by 15%denaturing polyacrylamide gelelectrophoresis.The strategy for the elaboration of the tDNA is summarized in Fig. 2. Cleavage of the tRNA and tDNA Substrates by Fe.Bleomycin "Reaction mixtures (5 pl total volume) contained 2-8 p~ (final nucleotide concentrations) radiolabeled tRNA or tDNA in 5 m~ sodium phosphate buffer, pH 7.5. The reactions were initiated by the simultaneous addition of freshly prepared solutions containing equal amounts of Fez+ and BLM Az to the final concentrations indicated in thefigure legends. Where additional components such as Mg2+ wereincluded, they were added to the reaction prior to Fez+ andBLM without subsequent renaturation of the RNA or DNA substrate. Reaction mixtures were incubated at room temperature for 15 min, then quenched by the addition of 3 pl of loading buffer (80% (w/v)formamide, 50 m~ Tris borate, pH 8.3, 1m~ EDTA, 0.1%(w/v) xylene cyanol, and 0.1% (w/v) bromphenolblue) and analyzed on denaturing 10%polyacrylamide gels (50 W for 1.5-2 h). Maxam-Gilbert DNA sequencing was carried out essentially as described (27). RESULTS
The tRNAHisprecursor substrate employed in thisstudy was prepared by in vitro transcription from an EcoRI-linearized DNA plasmid, as described previously (10-12, 25). The tDNA substrate was prepared by ligation of two synthetic DNAs 62 and 56 nt in length, respectively. The ligation was accomplished via the agency of T4 DNA ligase aRer the fragments had been aligned by annealing toa 27-nt DNA oligonucleotide complementary to the 3'- and 5"terminal sequences in the appropriate tDNA "fragments." The 118-nt product was then
5'
27nt
I
5'-GGCTCTGGCATTCGTGGGTI'CGATI'CCCATCAATCGCCCCAAATAAAAATTGAATT-3' 5'-GAATGCCAGAGCCACAATCTGGT-
3'
7
5'
OH -0,PO
1) 65%. 50mM NaCl 2) slow cooling
5'
3'
I
1) T4 DNA ligase 2) 15% PAGE
(31
FIG.2. SchemeemployedforelaborationofthetDNAsubstrate. T w o chemically synthesized tDNA fragments were5'-phos-
phorylated, then annealed to a 27-nt DNA oligonucleotide complementary to the 3'- and 5"terminal sequences in the 62- and 56-nt fragments, respectively. The fragments were then ligated with T4 DNA ligase to afford the requisite tDNA substrate.
purified by 15%polyacrylamide gel electrophoresis (Fig. 2). The ability of the tDNA to act as a substrate for oxidative cleavage by Fe.BLM was examined under the reaction conditions employedpreviouslyfortRNA (10-12, 14) (Fig. 3). Fe(II).BLM-dependent cleavage of the tDNA obtained at a BLM concentration as low as 250 rm in the absence of any added reducing agent, affording a single major cleavage band. This same band was also prominent when the substrate was treated with 500 IIM Fe(II).BLM,although bands resulting from cleavage at several additional sites were also apparent. Higher concentrations of Fe(1I)eBLM resulted in essentially complete disappearance of the intact tDNA substrate. In separate experiments under comparable conditions it was shown that the cleavage reaction was essentially complete within 2 min. Sequence analysis of the tDNA products revealed the major
~
Cleavage of tRNAHisPrecursor and tDNAHisby Fe-Bleomycin 1
3
5
7
9
1
1
1
3
5
7
9
25911 1 1 1 3 1 5
FIG. 4. Comparison of the cleavage of tRNAHi. and tDNAH* precursor substrates by Fe(II).BLM. The reactions were run as FIG. 3. Fe(II).bleomycin-mediatedcleavage DNAALprecursor described under "Experimental Procedures" using the tRNAtDNA and substrate. Reactions were run and analyzed as described under "Ex- substrates at 7-8 p~ nucleotide concentrations.Lane 1, tDNAonly; lane perimental Procedures." Lane 1, DNA alone (-2.1 p~ nucleotide con- 2, 1.25 p~ BLM AZ; lanes 3-8, 0.25, 0.5, 1.25, 2.5, and 250 25, p centration); lane 2,2.5p BLM; lane 3,2.5p~ Fez+;lanes 4-8,0.25,0.5, Fe(II).BLM Az, respectively; lane 9, tRNA only; lane 10, 1.25 p~ BLM 1.25, 2.5, and 5.0 p Fe(II).BLM, respectively; lane 9,G-lane; lane 10, A,; lanes 11-16, 0.25, 0.5, 1.25, 2.5, and 250 25, p Fe(II).BLM As, G + A lane; lane 11, C + T lane; lane 12, C lane. respectively.
cleavage site to be P5(Figs. 1 and 3), i.e. corresponding exactly 1 3 5 7 9 13 11 1519 17 21 to the major site of cleavage of the corresponding tRNA (Fig. 1) " (10-12, 14). In addition to this unexpected finding, BLM also GZ7,G37, A40, and P3) cleaved the tDNA at five sites (C23, corresponding to five of the six minor cleavage sites in the tRNAHis precursor substrate. The tDNA substrate was also cleaved at several additional sites that did not correspond to sites of cleavage in the tRNAHissubstrate. Interestingly, many of these sites were clustered at the junction between the acceptor stem and dihydrouridine stem-loop structures. While the basis for this selectivity is unclear, it may be noted that the majority of cleavage sites in the tRNAHissubstrate also occurred in this region. Relative Eficiencies of tRNA and tDNA Binding and Cleavage-The relative efficiencies of tRNA and tDNA cleavage by Fe(II).BLMA2 were evaluated using the two substrates at comparable concentrations.As illustrated in Fig. 4, cleavage of the tDNA was readily detectable using 500 n~ Fe(II).BLM, i.e. at a 15:l nuc1eotide:BLM ratio. Substantial cleavage of the tDNA substrate was apparent using 1.25 p~ Fe(I1)-BLM, and the at higher concentrationsof substrate was completely consumed FIG.5.The effects of unlabeled tRNAand tDNAonthe Fe-BL" Fe(II).BLM (lams 3-8). In contrast, cleavage of tRNAHis was mediated cleavage of B'-end labeled tRNAHi" and tDNAHinpredetectable in the presence of 1.25 p~ Fe(II).BLM, but proceeded cursors. The reactions were run as described under "Experimental Procedures" using radiolabeled tRNA and tDNA substrates at 7-8 p~ readily only at 2.5 p~ Fe(I1)-BLM.In contrast with the results nucleotide concentrations. Lane 1, radiolabeled tRNAH'" alone; lanes obtained for the tDNA, higher concentrationsof Fe(II).BLM did 2-6, radiolabeled tRNAH'"+ 25 p Fe(II).BLMA, + 0,4,8,16,and 80 p respectively; lanes 7-11, radiolabeled tRNAHi"+ 25 not produce significantlygreater cleavage of the tRNAHispre- unlabeled tDNAHB8, p Fe(II).BLM A, + 0,4, 8, and 16,80 p~ unlabeled tRNAHi",respeccursor substrate. tively; lane 12, radiolabeled tDNAHiBalone; lanes 13-17, radiolabeled To assess whether the relative efficiencies of tRNA and tDNA tDNAHiB + 1.25 p Fe(II).BLM Az + 0, 4, 8, 16,and 80 p~ unlabeled cleavage wererelated to the relative affinities of Fe(II).BLM for tDNAHia, respectively; lanes 18-22, radiolabeled tDNAHi" + 1.25 p~ the two substrates, competition experiments were carried out Fe(II).BLMAz + 0,4,8, 16,and 80 p~ unlabeled tRNAHi*,respectively. presence of using radiolabeled tRNA or tDNA substrates in the the unlabeled substrates. As shown in Fig. 5, the cleavage of up to 80 p ~ In . contrast, admixture of 80 p~ unlabeled tRNAHis radiolabeled tRNAHis (7-8 p~ nucleotide concentration) was to the labeled tRNA dramatically diminished cleavage of the little affected by unlabeled tDNA at any tested concentration labeled substrate. Analogously, cleavage of radiolabeled tDNA
Cleavage of tRNAHisPrecursor and tDNAHisby Fe-Bleomycin
25912
(7-8 w nucleotide concentration) by 1.25 1.1~Fe(II).BLM was inhibited by 80 w unlabeled tDNA, but greater inhibition of tDNA cleavage was obtained by admixture of unlabeled tRNAHiS.These data argue that Fe(II).BLM A2 is bound to tRNAHi" more strongly than to the corresponding tDNA. Effect of M p on tDNA and tRNA Cleavage-Previous studies have suggested that Fe(II).BLM-mediatedcleavage of some RNA substrates may be more readily inhibited by added Mg2' than cleavage of B-form DNA substrates (13, 14). In order to explore the basis for this effect, the cleavage of tRNAHisand tDNA substrates was studied in the presence of varying concentrations of M e . As shown in Table I, both substrates were cleaved more efficiently in the absence of Mg2' than in its presence. However, the diminution of cleavage was comparable for both substrates. Furthermore, cleavage of both substrates was readily apparent even at 5 m M e concentration. These data suggest that the ability of Mg2' to inhibit cleavage of a polynucleotide substrate by Fe(II).BLM is related primarily to the three-dimensional structure of thatsubstrate, not to whether it is constituted from ribonucleotides or deoxyribonucleotides. DISCUSSION
Earlier studiesof Fe(II).BLM-mediatedRNA strand scission indicated that RNA cleavage occurred in a more highly selective fashion than DNA cleavage. Furthermore, while 5' G-pyr 3' sites were well represented among RNA cleavage sites, as is also true for DNA, sites of RNA cleavage were notedfrequently at the junction between double- and single-stranded regions of the RNA substrate (10, 14). The last of these observations seemed to constitute a sharp distinction between the cleavage of RNAand DNA by BLM.Detailed analyses of DNAsubstrates amenable to cleavage by BLM indicated that normal DNA duplexes weregenerally good substrates, butthat single-stranded regions of DNAs (28,291 or Z-DNA (17) were not. These observations notwithstanding, it should also be noted that several studies have suggested that the recognition ofDNAbyBLM must also have a conformational component. For example, Haidle and co-workers (30) found that treatmentof linearized plasmid DNAs with a limited amount ofBLM resulted in small numbers of breaks a t discrete sites highly susceptible to cleavage. Likewise, Mirabelli et al. (31) compared the cleavage of Form I and Form I11 pBR322 DNAs and found that the supercoiled substrate was cleaved at some sites not affected in Form I11 pBR322 DNA. In an analogous fashion, local alteration of DNAconformation by platination (19,201, methylation (16-181, or the introduction of bulges (21) altered the pattern of DNA cleavage substantially. In view of the dependence of DNA cleavage patterns on the conformation of the individual substrates, it seemed likelythat TABLE I Effect of Mg2' on the cleavage of tRNAHk and tDNAHi' precursors by Fe(II).BLM Reactions were carried out as described under "Experimental Procedures" using radiolabeled tRNAHi' or tDNAHis substrates a t 7-8 nucleotide concentration and 25 m (for tRNA) or 1.25 m (for tDNA) Fe(II).BLM. Mg2+ concentration tRNAH's
Substrate tDNAH'" % cleavage
rnM
0 0.25 0.5 2.0 5.0
10.4 4.4
13.5 7.2 4.3 1.8 1.8
~~
Represents % conversion of total RNAor DNAtothe major cleavage product (U35pT35).Control reactions lacking Fez+ or BLM gave negligible (
