Bleomycin mediated degradationof DNA-RNA hybrids - BioMedSearch

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Bleomycin mediated degradationof DNA-RNA hybrids does not involve C-1' chemistry. M.J.Absalon, C.R.Krishnamoorthy1, G.McGall2, J.W.Kozarichl and ...
Nucleic Acids Research, Vol. 20, No. 16 4179-4185

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry M.J.Absalon, C.R.Krishnamoorthy1, G.McGall2, J.W.Kozarichl and J.Stubbe* Department of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, 'Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 and 2Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, CA 94304, USA Received June 4, 1992; Revised and Accepted July 18, 1992

ABSTRACT Incubation of Fe(ll) bleomycin and 02 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm 02 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to detemine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1 '-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1 '-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H20 nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1 '-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-i' chemistry, but a direct consequence of C-4' chemistry. INTRODUCTION Bleomycin (BLM) is an antitumor antibiotic whose cytotoxicity is thought to be related to its ability to bind to and degrade dsDNA (1,2,3). This degradative process requires two cofactors, a metal ion and 02, and results in the formation of two types of monomeric products, base propenal and nucleic acid base (4,5). Studies from our laboratories using specifically isotopically labeled DNAs have shown that both of these products result from the partitioning of a common 4'-radical intermediate subsequent to abstraction of the 4'-hydrogen atom by activated BLM (Scheme I) (6, 7). The sequence dependent isotope effects on these reactions vary from 1.5 to 6.0 (7). Extensive investigation of the mechanism of these reactions by numerous laboratories have shown that the base propenal pathway requires an additional molecule of 02 to that required to form activated BLM and results in strand scission under neutral conditions yielding DNA fragments containing 3'-phosphoglycolate ends and 5'-phosphate *

To whom correspondence should be addressed

ends (1, 2, 8-10). The pathway leading to nucleic acid base release, does not require additional 02 and results in formation of a 4'-keto-1 '-aldehyde sugar moiety in an intact strand which only after alkaline treatment results in strand scission and fragments with 3'-phosphate and 5'-phosphate ends (11-13). The major site of damage mediated by activated BLM clearly results from C-4' chemistry at the pyrimidine in dGT or dGC steps (14, 15). While C4' chemistry has been well defined, the question arises as to whether additional 'minor' chemistries can occur in the degradation of BLM by DNA. Our recent studies of the mechanism of ene diyne DNA cleavers using specifically isotopically labeled DNAs have revealed chemistry of minor lesions, not easily detectable by normal analytical methods (16-18). For example, the major lesion resulting from the interaction of neocarzinostatin (NCS) with DNA has been shown by Goldberg and his collaborators to occur at the 5' position of Ts and dAs (19). However, chemistry at minor lesions such as C-i' at the dC in d(AGC) sequences (16) and C4' at the T and d(GT) steps were easily detected, using the isotopic effect methodology we have developed (17, 18). Thus, chemistry of minor lesions, difficult to detect due to the unavailability of good analytical methods, deserves careful scrutiny as these minor lesions may play a significant role in the observed cytotoxicity. An additional recent observation made us consider likely the possibility that BLM might also be capable of alternative chemistry within the minor groove, effecting hydrogen atom abstraction from either C-i' or C-S' in addition to C-4' (20). Our studies of the interaction of BLM with DNA-RNA hybrids, specifically poly(dA) poly(rU), with FeBLM at 4°C and 4 atm 1.0. 02 yielded a ratio of adenine propenal: adenine of Similar studies with generic B-form DNA typically give ratios of 10, demonstrating that high concentrations of 02 shift partitioning of the 4'-radical intermediate almost exclusively to pathway Aa, Scheme I. While the structures of poly(dA) poly(rU) and DNA-RNA hybrids in general are still controversial; the general concensus is that while the individual strands of the heteroduplex retain their conformations observed in nonhybrids, the overall conformation is more 'A'-like having a shallower and wider minor groove (21-25). In this conformation both the 1' -

-

4180 Nucleic Acids Research, Vol. 20, No. 16 and 4'-hydrogens are potentially accessible to attack by activated BLM. Cleavage of the C-i' carbon hydrogen bond (pathway B, Scheme I) followed by an 02 dependent or an independent pathway, in analogy with C4' chemistry (Scheme I), would lead in both cases to nucleic acid base release and hence might account for the inability of 02 to suppress adenine release from poly(dA) poly(rU) (26, 27). The present communication examines the possibility that 'activated BLM' can catalyze C-i' carbon-hydrogen bond cleavage in ds-DNA or in DNA-RNA hybrids. In the latter case, our hypothesis that C-i' chemistry can account for alternative product distribution was tested using poly([1 '-3H]dA) poly(rU), poly([l '-2H]dA) poly(rU) and poly([4'-2H]dA) poly(rU). Evidence is presented that while 'A' like polymers have altered product distribution from 'B-like' polymers, BLM mediated C-i' chemistry does not provide an explanation for these results. In addition, the use of [1'-2H]dC in defined ds-DNA sequences, suggests that C-i' chemistry does not occur.

EXPERIMENTAL Materials and Methods Poly(dA) * poly(dT); poly d(G-C) * poly d(G-C); poly(rA) * poly (dT); poly d(I-C) poly d(I-C) and poly (dI) poly(dC), poly(rU), PI nuclease, and phosphodiesterase I were purchased from P-L Biochemicals. Other DNA-RNA hybrids were prepared employing appropriate homopolymers obtained from P-L Biochemicals using known procedures (P-L Biochemicals, Product Reference Guide, 1984). Poly (dG) poly(dC), inorganic pyrophosphatase, E.coli alkaline phosphatase, and SI nuclease were obtained from Sigma Chemicals. Bleomycin (Blenoxane) was a gift from Bristol Laboratories. Ferrous ammonium sulfate, ferrous sulfate and 2-thiobarbituric acid, were obtained from Fisher Scientific. Nucleic acid bases were obtained from Fluka. Base propenals were synthesized following the procedure of Johnson et al. (1984) (28). [U-14C]ATP (specific activity 593 mCi/mmol was purchased from New England Nuclear-Dupont. 2-Deoxyribonolactone was synthesized according to literature procedures (29, 30). Terminal deoxynucleotidyl transferase (TdT) (specific activity 104-2.5 x 104 units/mg) was a generous gift of Dr. Mary Sue Coleman, University of Kentucky. Alternatively it was purchased from BRL Biochemical Corp. lot # 270730. d(CGCGCG) was a gift from John Gerlt, University of Maryland, and (dG)gdC was prepared using a Biosearch Milligen 8600 DNA synthesizer and standard procedures. All other chemicals were of reagent grade. Reaction Conditions for the interaction of BLM with DNA and DNA-RNA hybrids The following reaction conditions were adopted to assess the effect of 02 concentration on the partitioning of the monomeric products generated in these reactions. 1. Reaction at room temperature or at 00 and normal atmosphere. The details of the reaction mixture and analyses are identical to those previously described (20). 2. Reactions at 0°C with 02 bubbling at increased pressure. Reaction conditions were identical to those previously described (20) but the reaction vessels were maintained in a modified Amicon filtration chamber which allowed °2 bubUing at moderate rate through a 1.5 inch 22 gauge hypodermic needle

and incremental addition of Fe(ll) to the reaction mixture under elevated pressure of 4 atm at 0°C. As a control, authentic samples of appropriate monomeric products were incubated with Fe(II) and BLM under identical increased 02 concentrations and the stabilities of these compounds were verified by HPLC analysis.

Identification and quantitation of reactions products UV absorbance of the eluate from a reverse phase HPLC column was continuously monitored at multiple wavelengths using a flow cell mounted in a Hewlett-Packard 8450A fixed diode array spectrophotometer. All analyses were based on 3-4 experiments and the product identification was confirmed by coinjection of standards. Quantitation was determined by one of two methods: 1. Isolation of the each peak of material eluted from the HPLC, measurement of absorbance and use of known extinction coefficients 2. Preparation of a standard curve with defined amounts of material, using peak heights or peak areas. In the case of base propenals, quantitation of malondialdehyde was also carried out using the thiobarbituric acid assay; Xmax 532 nm, e = 1.6x i05 M'cm-' (31).

Synthesis of poly([1'-3H]dA) In a final volume of 500 ,uL was incubated 400 mM potassium cacodylate (pH 7.4), 32 mM MgCl2, 1.2 mM ZnCl2, 10 mM DTT, 10 1mol dG(dA)g, 0.04 mg BSA, 1.0 mM [1'-3HIdATP (specific activity 3.5 x 105 cpm/4mol), 15 units of TdT(BRL). The reaction mixture was incubated for 16 h at 37°. The reaction went to -75% completion as determined by both acid and ethanol precipitable radioactivity. The DNA was isolated by extraction with 4 x500 AL volumes of phenol saturated with 100 mM Tris(pH 8.0). The solution was then back extracted once with 100 mM Tris (pH 8.0) and the volume reduced to 100 AL by extraction with 2-butanol. The solution was then made 0.25 M in NaAc and the DNA was precipitated with two volumes of ethanol. After the pellet was washed, it was redissolved in 100 liL of 50 mM potassium phosphate (pH 7.0). Product analysis of BLM mediated degradation of poly([1'-3H]dA) poly(rU) A 150 yL solution of 1.0 mM poly([ 1_'-3H]dA) (specific activity = 3.5 x 105 cpm/4mol), 1.0 mM poly(rU) in 40 mM potassium phosphate (pH 7.6) was preincubated with 620 AM BLM at 4°C under 4 atm of 02 as described above. The reaction was initiated by the addition of 45 ytL of 5 mM ferrous ammonium sulphate and the incubation allowed to continue for an additional 30 min. At this time, a 5 FL aliquot from the reaction mixture was diluted with 395 ytL of a saturated TBA solution and assayed for total 'malondialdehyde-like' products (31). A 30 AL aliquot of the reaction mixture was adjusted to pH 5.5 with acetic acid and to 0.4 mM Zn2+ with ZnCl2 and digested with 2 U of P1 nuclease in a final volume of 50 AL. The reaction mixture was incubated at 370 for 45 min. The products of the reaction mixture were analyzed by reverse phase HPLC. The products were eluted for 5 min with 5 mM NH4Ac (pH 5.5) followed by a linear gradient from 0 to 30% CH30H between 5 and 30 min, followed by isocratic elution at 30% CH30H for an additional 15 min. Compound, specific activity, retention time: UMP, 2 min; AMP, 3.5 x 105 cpm/4mol, 7 min; adenine, 20.5 min; adenine propenal, 3.5 x 105 cpm/4mol, 30.5 min. The remaining 160 IL of the reaction was diluted with 440 yL H20 and the water and volatile products isolated by bulb-to-bulb distillation. The distillate was made 50 mM in potassium hydroxide and again

Nucleic Acids Research, Vol. 20, No. 16 4181

subjected to distillation. Tritium from the first and second distillate were combined and quantitated by liquid scintillation counting.

Synthesis of [1'-3H and 2H]adenosine [1 -3H]-1-Acetyl-2,3,5-tribenzoylated ribose (ATBR) was synthesized by a modification of the procedure reported by Kohn et al. (32). Details of this procedure are available on request. The resulting product was recyrstallized from 5 mL ethanol at -20°C yielding 390 mg (46% yield with a specific activity 1.Ox 107 cpm/4mol). The [1-3H]ATBR, diluted to have a specific activity of 106 cpm ltmol- , was charged with the purine base, adenine, using a modification of the procedure reported by Vorbruggen and Bennua (33). The resulting nucleoside was recrystallized from methanol at - 20°C yielding 78 mg (62 % yield) of [1'-3H]adenosine (specific activity 106 cpm ,umol-1). The 'H NMR was identical to commercially available adenosine. The [1'-2H]adenosine was prepared by an analogous procedure. The monophosphorylations of 1 '-2H, 1 '-3H and 4'-2H adenosine were carried out by the procedure of Yoshikawa et al. (34) Yields of the corresponding isotopically labeled AMP typically ranged from 65 to 90%. Conversion to the corresponding triphosphate were carried out by the procedure of Hoard and Ott (35) giving the desired product in 35 to 75% yield.

Synthesis of [4'-2H]adenosine [4'-2H]Adenosine was prepared as previously described (6). The nucleoside was converted to the triphosphate by standard procedures (34, 35). NMR analysis revealed 4'-2H incorporation to greater than 95%. Conversion of ATP to dATP Reduction of [U-14C]ATP, [1'-2H]ATP, [1'-3H]ATP, [1'-2H] CTP to the appropriate dNTP was accomplished using Lactobacillus leichmannii ribonucleoside triphosphate reductase (RTPR) by previously described procedures (36).

Preparation of poly(dA) Labeled deoxynucleotides with the exception of the 1'-3H material, were polymerized using calf thymus TdT provided by Dr. Coleman. In a typical reaction TdT (160 units) was added to a 100 AL of 200 mM potassium cacadylate (pH 7.5) containing 18 mM labeled dATP, 0.16 mM (dG)gdC primer, 8 mM MgCl2, 1 mM 3-mercaptoethanol, 1.7 units pyrophosphatase and incubated for 1-2 days. Progress of the reaction was monitored by following the change in absorbance at 260 nm of a 1.0-/,L aliquot added to 400 /L of 50 mM potassium phosphate (pH 7.0). Polymerization was considered 100% complete when the absorbance at 260 nm approached 55 % of the initial reading. The polymerization reaction was quenched with 10 mM EDTA when no additional change in absorbance at 260 nm occurred. The crude poly(dA) was isolated as described under synthesis of poly([1 '-3H]dA). Nucleotide concentration was determined using e = 8.6x 103 M-Icm-1. Yields typically were 50% based on the amount of nucleotide. Selection effect on adenine and adenine propenal formation when poly([4'-2H]dA) poly(rU) interacts with activated BLM Degradation of poly([1'-'H]dA) poly(rU) occurred in a total volume of 100 AL containing 660 AtM poly([U-14C]dA) (specific activity 1.4x 105 cpm ,Amol-1), 660 jAM poly(rU), 600 jiM of

carrier oligonucleotide d(CGCGCG)2, 20 mM HEPES (pH 7.5), 20 mM NaCl, and 400 jiM BLM. The solution was allowed to incubate for 10 min at 4°C under 4 atm 02 and the reaction initiated by the addition of 600 AM ferrous ammonium sulfate. The reaction was stirred at 4°C under 4 atm of 02 for an additional 10 min at which point the products of the entire reaction were separated by HPLC using an Econosil C-18 HPLC column. The compounds were eluted at 1 mL min-I using a linear gradient from 0 to 20% methanol over 20 min, followed by isocratic elution at 20% CH30H for 10 min followed by a linear gradient from 20 to 50% methanol over 20 min. Adenine propenal (retention time, 31 min) was collected and quantified using the TBA assay and by scintillation counting. Analysis of the degradation products from the poly([4'-2H]dA) poly(rU) reaction mixture was similar to that of the [4'-JH] polymer except that the reaction solution contained 600 AM poly([4'-2H]dA) and 60 ,iM poly([U-14C]dA), specific activity 1.4 x 106 cpm Amol*-l. The selection effect was determined by the normalized ratio of adenine propenal from the [4'-'H] to the amount produced from the poly([4'-2H]dA) after the relative contribution of base propenal from the proteated material as determined from the carbon-14 sample was subtracted from the absolute amount measured in the TBA assay.

Determination of the adenine: adenine propenal ratio at elevated 02 concentrations with poly([4'-2H]dA) poly(rU) A typical reaction contained in a final volume of 83 jL, 1.2 mM of either the poly([4'-1H] or [4'-2H]dA), 1.2 mM poly(rU), 0.40 mM BLM, 0.6 mM ferrous ammonium sulfate in 20 mM HEPES (pH 7.5). Poly(dA) and poly(rU) were mixed at 65°C for 5 min and allowed to cool slowly to room temperature before being cooled on ice. Reactions were carried out in a 1.5 mL Eppendorf tube equipped with a magnetic stir vane under 4 atm 02 at 4°C as previously described. Reactions were initiated after 10 min by the addition of the freshly prepared ferrous ammonium sulfate and incubated at 4°C and 4 atm 02 with stirring for 35 min. Completed reactions were immediately diluted with 1.0 mL cold H20. A 100-jL aliquot of this diluted mixture was used to determine 'total malondialdehyde like products' (31). A 200 ,uL aliquot was used for analysis by HPLC to determine the amount of adenine released and its ratio to the amount of adenine propenal released. An Econosil 10 ,i column (250 mmx4.5 cm), flow rate, 2 mL min- , was used. The products were eluted using a linear gradient from 0 to 10% CH3 OH over 5 min; isocratic elution at 10% methanol for 5 min followed by a linear gradient from 10 to 20% methanol over 5 min, isocratic elution at 20% CH30H for 20 min followed by a linear gradient from 20-40% methanol over 10 min. Ammonium acetate (5 mM, pH 5.5) was used as the aqueous buffer, compounds were detected, and quantitated as described above.

Degradation of poly(dA) poly(rU) by BLM and subsequent enzymatic digestion to isolate deoxyribonolactone BLM reactions were carried out as indicated above with the exceptions that poly([U-'4C]dA) [specific activity 2.0x 106 cpm Amol-lJ was used in place of the non-radioactive DNA strand and the reactions were scaled up to 500 jiL final volume. An 88 AL-aliquot of the reaction mixture was used for analysis by HPLC. Adenine and adenine propenal were collected from the HPLC and quantified as described above. NaOAc (38 ,uL, 4 M) and polyrU (70 jiL, 5 mM) as carrier was added to a 400 jiL aliquot of the reaction mixture and the hybrid precipitated with

4182 Nucleic Acids Research, Vol. 20, No. 16 1.5 mL ethanol at -20°C for 6 h. The pelleted DNA was digested with S I nuclease, alkaline phosphatase and phosphodiesterase, as previously described (26). Prior to separation of the monomeric products produced from this digest by HPLC, 2-deoxyribonolactone (100 itmol) was added as carrier and the reaction mixture analyzed by reverse phase HPLC. An Econosil u C18 column (250 x4.5 mm) was used: flow rate, 1 mL min'-l. The products were eluted using a linear gradient from 0-15% CH30H, duration five min followed by a linear gradient from 15 to 50% CH30H over 20 min; Ammonium acetate (5 mM, pH 5.5) was used as the aqueous buffer. Fractions were collected that eluted from 2-6 min, and 6-10 min, as were the peaks containing uridine (12 min), adenine (16.2 min) and deoxyadenosine (22.5 min). Each fraction was analyzed for radioactivity. The void volume fraction (2 -6 min) (25.6 x 103 cpm) was concentrated to dryness and redissolved in 150 uL 0.01 N HCl and heated at 90°C for 15 min. Two 40-p,l aliquots (6,840 cpm) were loaded evenly across a TLC silica plate (20 x 20 cm) and developed as described in Fig. 1A and lB. Each plate was divided into 1 cm longitudinal sections and the silica scraped into scintillation vials before 0.5 mL of 1 M HCI was added followed by 8 mL of scintillation fluid. The contents were analyzed for radioactivity by scintillation counting and the rf values compared to authentic deoxyribonolactone and glycolic acid standards.

A

a

1000

IL) (lactone standard migrates here)

11, o _ 0.0

1, 0

0s

0.

Rf

B

3000,

RESULTS AND DISCUSSION 0.

Incubation of [1'-2H]dC-DNA with activated BLM Studies using [4'-2H]dN-DNAs revealed isotope effects varying from 1.5 to 6.0 on DNA strand scission mediated by BLM (7). Specifically isotopically labeled DNAs have also recently been used to detect the chemistry of minor lesions in NCS mediated DNA degradation (16-18). In an effort to determine if C-i' chemistry could contribute to a minor extent to the observed DNA damage mediated by activated BLM, we prepared [1'-2H]dC which was incorporated into a variety of DNA fragments. No evidence for C-i' chemistry in a variety of sequence contexts was observed. Isotope effects of 1.2 to 1.3 are readily detected by this method. If C-i' chemistry does occur, in contrast to C4' chemistry, there are no observable isotope effects on the cleavage. In addition, C-i' labeling does not appear to enhance the cleavage at C4' as might be anticipated if intramolecular partitioning was occurring.

Analysis of product distribution using DNA-RNA homopolymer hybrids As we recently reported using poly(dA) poly(rU), a 'putative Alike' polymer, incubated with activated BLM resulted in a markedly perturbed base propenal to base ratio at high 02 concentrations in comparison with similar reactions carried out on generic 'B' form DNA (20). We suggested at that time that the large amount of free base release observed might be explained if BLM mediated chemistry at the C-i' position in addition to the C4' chemistry observed in ds-DNA. This hypothesis was based on the fact that the minor groove of 'A-like' molecules is both shallower and wider than 'B-like' molecules potentially facilitating access of activated BLM to the C-i' hydrogen. To determine if the ratio of base propenal: base can be altered as a finction of DNA conformation and 02 concentration, a variety of additional homopolymers, both ds DNA and DNA-RNA hybrids were examined. The results are summarized in Table I. Only two of the homopolymers examined, poly(dI) poly(rC)

2000'

(lactone standard migrates here) 1000

_~~~~~~iIF1 n. 0.0

02

0

0.4

0.

1.0

Rf

Fig. 1. TLC-Separation of Deoxyribonolactone from other Products of BLM Mediated Degradation of poly([U-14C]dA poly(rU). (A) Silica gel TLC plate developed in a solution of methylethyl ketone, ethanol and triethylamine (35:15:2 VNNV). Carrier deoxyribonolactone migrates with an Rf -0.4 to 0.45 as indicated by the arrows. Lactone carrier was visualized as previously described (37). Glycolate (Rf = 0.2). (B) Silica gel TLC plate developed in a solution of methyl ethyl ketone, isopropanol, triethylamine (10:10:1 VNVNV). Carrier deoxyribose lactone migrates with an Rf value of 0.35-0.40 as indicated by the arrows. Glycolate (Rf = 0).

Table I. Interaction of DNA-RNA hybrids and DNA with activated BLM at 4 atm

02

Substrate

Relative Ratios base:base propenal Air 4 atM

poly(dA) poly(dT) poly(rA) poly(dT) poly(dA) poly(rU) poly(rA) poly(dU) poly(dGdC) poly(dG) poly(dC) poly(dIdC) poly(dI) poly(dC) poly(dI) poly(rC) poly(rI) poly(dC)

1:1 1:1 3:1 1:1 2:1 2:1 2:1 1:1 2:1 2:1

B*

=

more

B-like features

Putative Conformation 02

21:10 1:6 1:1 1:6 21:10 1:6 21:10 > 1:10 1:2 >1:10

B B* A B B B* B B A B

Nucleic Acids Research, Vol. 20, No. 16 4183 Table H. Quantitation of products from the reaction of Poly([1 '-3H]dA) poly(dU) with activated BLM Substrate

Reaction scale (nmol)

total TBA positive products (nmol)

Adenine tHPLCI (nmol)

Adenine-propenal tHPLCJ (nmol)

Volatile tritium (nmol)

150

14.7

13.2

12

1.5

15.3

9.0

1.6

Exp I

poly((l' 3H]dA)a poly(rU)

Exp 2 a

150

aThe experiment was carried out 5 times. The data indicated are representative results.

Table HI. Quantitation of the adenine to adenine propenal ratio upon 1' or 4'-Deuteriation of polydA in poly(dA)poly(rU) Substrate

Reaction scale (nmol)

poly([4'-2H]dA)

100

5.14

poly(dA) poly(rU)

100

7.74

poly( 1'-2H]dA) poly(rU)

160

14.0

Adenine IHPLCJ (nmol)

total TBA positive products (nmol)

Adenine propenal/ Adenine HPLC*

TBA-positive products/ Adenine

4.0

1.12

1.3

5.7

1.14

1.4

_

1.1

poly(rU)

12.5t

*Quantitated from the A260 of the collected HPLC fraction using e = 13.7 x 103 M- Icm-I tCalculated by comparing the area of the respective peaks after correcting for the difference in extinction coefficients for the absorbance at 260 nm. These experiments were each carried out twice.

and poly(dA) poly(rU), had base propenal ratios of

1.0 at high 02 concentrations. Both of these polymers are reported to have 'A-like' conformations (21, 38). Interestingly poly(rI) poly(dC) has been reported to have more 'B-like' characteristics consistent with the base propenal to base ratio (at 4 atm 02) of 10. While poly(rA) poly(dT) has been postulated by many investigators to be 'A-like', recent structural studies of Gupta et al. (39) and Kuregar et al. (40) suggest that it is B-like. These results are again consistent with the observed ratio of 10 of base propenal to base. A second interesting observation made using the DNA-RNA hybrids was that in no case were products observed from degradation of the RNA strand (data not shown), a result consistent with earlier studies by Haidle and Bearden (1975) (41). While recent studies of Magliozzo et al. (1984) (42) and Carter et al. (1990 and 1991) (43, 44) have suggested that on a rare occasion one can observe very efficient cleavage of a tRNA molecule, still no examples exist where the RNA is 'cleaved' in a hybrid structure. The structural implications of these observations must be incorporated into any model of the binding to and reaction of activated BLM with DNA. -

4'-11 abstractio

I'-H abstraction

0

-

Does C-1' chemistry account for altered base propenal to base ratios? The studies on the homopolymer hybrids suggested that the model invoking C- ' chemistry to accommodate altered base formation is a viable one. Three experiments were therefore designed to test this hypothesis. Incubation of poly([1'-3H]dA) poly(rU) with activated BLM Our model (Scheme I) predicts that incubation of poly([1'-3H]dA) poly(rU) with BLM should result in 3H20 production concomitant with adenine release. Poly([1'-3H]dA)

>

\

additional

0

02I O=P-O-R'

O=P-O-R-

02

R-O-P-0

I1-

0

Pathway R

O0

Pathwlag B

s

O

R-0-P-0

-0-P-0

A

A

0

HO)_

0=P-0-

0 O=P-0-R-

I-

O=P-0-R' 0I-

0

0

.0_

-

o 0 R-O-P-O

R-0-P-O

0

R-O-P-0

I

-

O'

0

0

A

A

O=P-O-R0

0

+

O=P-0-RI-

0

Rdenine

Rdenine

Scheme 1

4184 Nucleic Acids Research, Vol. 20, No. 16 poly(rU) was prepared and the amount of adenine and 3H20 released under 4 atm 02 was determined. As indicated in Table II, for every molecule of adenine released at best 0.1 equivalent of 3H20 was produced. There are two possible explanations to accommodate these results. The first is that the C-i' carbon hydrogen bond is cleaved and that there is a very large isotope effect on this cleavage. Precedent for this interpretation comes from our original studies using a dsDNA polydA [4'-3H]dU in which a global selection effect of 10 is observed on the cleavage of the 4' carbon hydrogen bond (6). However, the specific activity of the [1'-3H]dAMP recovered at the end of the reaction subsequent to digestion with P. nuclease was identical to that of the starting material seeming to argue against this interpretation. In addition, for reasons we describe below, an alternative interpretation of these results is favored. During this reaction [1 '-3H]adenine propenal is also generated. This molecule can decompose (to a small extent) to produce [3H]-malondialdehdye which is also volatile. Thus we attribute the small amounts of volatile 3H observed in these reactions to breakdown of the adenine propenal and not to 3H20. Addition of KOH to make the enolate of malondialdehyde prior to the analysis for volatile 3H, reduces the volatile 3H an additional two to three fold. Several additional lines of evidences described subsequently support the conclusion from these studies that the C-i' carbon hydrogen bond is not cleaved during degradation of DNA by activated BLM. -

Attempts to identify deoxyribonolactone Studies of Kappen et al. with NCS (26) and Sigman et al. (27) with copper o-phenanthroline indicate that hydrogen atom abstraction from C-i' ( ±fi 02) of a deoxyribose moiety of DNA by these DNA cleavers results in production of an intact DNA strand containing deoxyribonolactone and its breakdown product. Methods to isolate this lactone in high yield from the intact DNA have been developed (26). Our model predicts that deoxyribonolactone should be produced stoichiometrically with base during this reaction. It should be noted however that the mechanism by which this product is produced, subsequent to 1' hydrogen abstraction and trapping by 02 is still obscure. Poly([U-14C]dA) poly(rU) was incubated with activated BLM under 4 atm 02 and the amount of adenine and adenine propenal quantitated. The polymer was then subjected to analysis similar to that described by Kappen et al. (26) and subsequent to enzymatic digestion and HPLC chromatography analyzed by TLC (Fig. IA, iB) for deoxyribonolactone. The Rf value of glycolic acid, the other major degradation product produced from the 3' phosphoglycolate ends accompanying adenine propenal formation, is clearly separated from deoxyribonolactone. In the second solvent system, no lactone is detected. These data indicate that for every equivalent of adenine less than 0.01 equivalent of deoxyribonolactone is produced. These results suggest that chemistry at C-i' is not occurring. Use of poly([4'-2H]dA) poly(rU) and poly([ 1 '-2H]dA) poly(rU) to detect C-i' chemistry The most compelling evidence against BLM mediated C-i' chemistry are the results from our studies using specifically deuteriated poly(dA)poly(rU). If the results from the experiments using poly([ 1_'-3H]dA) poly(rU) are actually the consequence of a large isotope effect on cleavage of the carbon-hydrogen bond

C-i', a large deuterium isotope effect, on the order of 4.9 by the Swain-Schaad relationship (45), should result from studies at

using poly([ 1-2H]dA) poly(rU). These results should be manifested in a shift from C-1' to C4' chemistry, intramolecular discrimination, and at high 02 concentrations should result in a dramatic increase in the adenine propenal to adenine ratio. Alternatively, incubation of poly([4'-2H]dA) poly(rU) with BLM should again result in an intramolecular discrimination against C4' hydrogen atom abstraction to favor C- ' hydrogen atom abstraction. These results would be manifested in an even smaller (