Bich T. N. Vu 1, Yinsheng Wang 2, Vairamani Mariappanadar 3, John-Stephen A. Taylor 1 and Michael L. Gross 1

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1 Study of Deamination Kinetics of 5-Methyl Cytosine-Containing Pyrimidine Dimers in ligodeoxynucleotides by uclease P1 Enzyme Digestion Coupled with Tandem Mass Spectrometry Bich T.. Vu 1, Yinsheng Wang 2, Vairamani Mariappanadar 3, John-Stephen A. Taylor 1 and Michael L. Gross 1 1) Washington University in St. Louis, Dept. of Chemistry, St. Louis, M 2) University of California at Riverside, Dept. of Chemistry, Riverside, CA 3) Indian Institute of Chemical Technology, Hyderabad, India

2 Purpose To study the deamination kinetics of 5-methylcytosine in oligodeoxynucleotides (Ds) containing cis-syn cyclobutane pyrimidine dimers (CPDs) photodamage. Methods A quantitative nuclease P1 enzyme coupled with tandem mass spectrometry method was implemented to determine the deamination rate. Results The deamination rates of 5-methylated cytosines in CPDs were obtained in single-strand and duplex Ds, allowing us to begin to explore systematically the effects of sequence context, flanking sequence, and secondary structure on the rates of this important reaction.

3 The majority of mutations that cause skin cancer are C T transitions or CC TT mutations at dipyrimidine sequences resulting from UV-lightinduced cyclobutane pyrimidine dimer (CPD) formation. Methylation of cytosine enhances by 5-15-fold pyrimidine-dimer formation, resulting from exposure of cells to sunlight. The enhancement may be due to the higher UV absorptivity of 5-methylcytosine vs cytosine in DA. The proposed mechanism for C-to-T transition mutations is the spontaneous deamination of methylated cytosines or cytosines. Clearly, the rate of deamination needs to be evaluated to define the role of 5- methylcytosines as a mutagen. Previously, 32 P postlabeling was employed to quantify DA photoproduct formation. We wish to continue to implement and apply uclease P1 enzyme-coupled ESI MS/MS as a quantitative method to study the deamination kinetics of 5-methylated cytosines in CPDs.

4 Photoproducts of DA at Dipyrimidine and TA Sites H 2 H 2 H 2 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 H H H 5' H H 3' 5' H [cis,syn] [trans,syn_i] [trans,syn_ii] UVB ( nm) UVB H 3' 5' H H 3' 5 TA mct 3 UVB/C ( nm) UVB TA* or T[ ]A H 5' CH 3 H H 2 3' H 2CH3 5' H H 3 C H [6,4] UVB/A ( nm) 3' H 2CH3 5' H H [Dewar] H 3 C 3' H

5 Deamination of 5-methylated Cytosine in UV-irradiated DA H 2 CH 3 CH 3 CH 3 CH 3 H H 2 H H H H 5' 3' 5 mc[c,s]t 3 H H 5' 3' 5 T[c,s]T 3 H 2 CH 3 CH 3 CH 3 CH 3 H H 2 H H H H 5' 3' 5 T[c,s]mC 3 H H 5' 3' 5 T[c,s] T 3

6 C T and CC TT Mutation Processes Error-prone replication 5 AA 3 3 mc T 5 Replication 5 GA 3 3 mct 5 UVB 5 GA 3 3 mc T 5 5 AA 3 3 TT 5 Deamination 5 GA 3 3 T T 5 DA pol. 5 AA 3 3 T T 5 Replication

7 uclease P1 enzyme from Penicillium citrinum has been used to degrade DA. It hydrolyzes single-strand DA to 5 - mononucleotide, and photodamaged DA to trinucleotides. For undamaged DA and Ds, the base stacks with phenyl group in the active site of the enzyme, and the hydrolysis proceeds. The photodamaged site of DA can not fit in the active site. The different products produced by nuclease P1 can be assigned by using MS on the basis of their molecular weight. The type of photoproducts can be identified by MS/MS.

8 The Mechanism of uclease P1 Cleavage uclease P1 enzyme cleaves undamaged DA to mononucleotide and damaged DA to trinucleotides. d(gt[ ]ATTAT) base dg, d(pt[ ]AT),d(pT), d(pa) Enz Phe P H

9 Materials Ds were obtained from IDT (Integrated DA Technologies). Duplex hairpins were annealed by heating 1-mM solutions of Ds to 90 C prior to use. Sample Preparations Single-strand and double-stranded Ds containing cis-syn dimers were prepared by irradiating the starting D with 450-W mercury arc light ~ 300nm after filtration). The solutions containing cis-syn dimers of D were incubated in 10-mM ammonium acetate buffer (ph = 6.8 at T = 25 C). Deamination reactions were carried out at T = 37 C as a function of time to obtain rate constants. Analysis The deaminated samples were digested with nuclease P1 for 3 min at RT. The solution resulting from nuclease P1 digestion was analyzed by ESI and MS/MS (Finnigan LCQ Classic). MS/MS data were acquired on the selected [M - H] - ions. To select both the deaminated and undeaminated components for fragmentation, the mass width for precursor selection was set at 5-6 m/z units.

10 uclease P1 Enzyme Coupled Tandem Mass Spectrometry Assay Identifying and quantifying photoproduct formations Deamination T mc T T P1 digestion P1 digestion dp, dpt mc dp, dpt T ESI coupled MS/MS ESI coupled MS/MS dpt mc dpt T m/z 802 m/z 803 The fragments of m/z 802 and 803 are specific for the two products. Their intensities were used to determine the fractions of undeaminated and deaminated components. m/z

11 The five photoproducts, [cys,syn], [trans,syn_ I], [trans,syn_ii], [6,4], [Dewar], after digestion with nuclease P1, give common products p[ ] that have the same m/z. Fortunately, the tandem mass spectra of these photomodified-containing trinucleotides are distinctive. The product-ion spectra of [trans,syn] isomers are different from that of [cis,syn] isomer by a fragment ion of m/z 745. [trans,syn_i] and [trans,syn_ii] isomers can be distinguished by a fragment ion of m/z 840. The [6-4] and [Dewar] isomers are different from CPD isomers by a loss of 113 u. The Dewar isomer produces a fragment of m/z 749, whereas the [6-4] isomer does not. In the case of TA photoproduct, it can be distinguished from other photoproducts by a characteristic fragments of m/z 616.

12 MS/MS of ESI-produced [M - H] - ions of uclease P1 Digestion Product for the cis-syn CPD Characteristic fragments of cis-syn dimers CACGTGmC[c,s]TGGCACCAC uclease P1 digestion d(pmc[c,s]tg) [M H] m/z 953 Deamination CACGTGT[c,s]TGGCACCAC uclease P1 digestion d(pt [c,s]tg) [M H] m/z 954 Relative Abundance [pt[c,s]pt H 2 ] [M G] [M G H 2 HP 3 ] [M H] m/z 1000

13 MS/MS of ESI-produced [M - H] - ions of uclease P1 Digestion Product for the trans-syn CPD Characteristic fragments of trans-syn dimers d(gagtat[t,s_i]tatgag) uclease P1 digestion d(pt[t,s_i]ta) [M H] m/z 938 Rel. Abund. [M H] m/z m/z d(gagtat[t,s_ii]tatgag) uclease P1 digestion d(pt[t,s_ii]ta) [M H] m/z 938 Rel. Abund. [M H] m/z m/z

14 MS/MS of ESI-produced [M - H] - ions of uclease P1 Digestion Product for the 6-4 and Dewar Photoproducts Characteristic fragments of 6-4 and Dewar dimers d(gtat[6-4]tat) uclease P1 digestion d(pt[6-4]ta) [M H] m/z 938 d(gtat[dewar]tat) uclease P1 digestion d(t[dewar]ta) [M H] m/z 938 Rel. Abund. Rel. Abund. [M + a 2H] m/z m/z [M + a 2H] m/z m/z

15 MS/MS of ESI-produced [M - H] - ions of uclease P1 Digestion Product for the TA Photoproduct Characteristic fragments of TA photoproduct GAGT[ ]ATmCATGAG uclease P1 digestion d(pt[ ]AT) [M H] m/z 938 Relative Abundance 616 [ptpa H 2 ] [M H] m/z m/z

16 MS/MS of ion of m/z 803, viewed in Zoom- Scan at Various Time Points of Deamination 803 dpt[c,s]t H dpmc[c,s]t- H 802 Relative Abundance 120 min 0 min m/z min 240 min 1800 min The peak of m/z 803 corresponding to deaminated component increases when deamination time increases, while the peak of m/z 802 corresponding to undeaminated component decreases.

17 Evaluation of the 1st rder Rate Constant from MS Data GTGmC[c,s]TGGCAC k = min -1 The fractions of deaminated and undeaminated species in the mixtures were calculated from peak areas obtained by integrating the peaks in the ion chromatogram after correcting for contributions from isotopic abundances. ln(%undeaminated) y = x R 2 = Hairpin CACGTGmC[c,s]TGGCACCAC k = min -1 y = x R 2 = Time (min)

18 Kinetic Parameters for 5-Methylcytosine Deamination in Cyclobutane Pyrimidine Dimers 37 C k (min -1 ) t 1/2 (min) GAGTAmC[c,s]TATGAG % 750 Flanking Adenines GAGTAT[c,s]mCATGAG Hairpin GCGCGTAmC[c,s]TATCGCG % Hairpin GCGCGTAT[c,s]mCATCGCG % 2600 Flanking Guanines GTGmC[c,s]TGGCAC GTGT[c,s]mCGGCAC Hairpin CACGTGmC[c,s]TGGCACCAC Hairpin GTGT[c,s]mCGGCACCAC % % % %

19 Duplex formation inhibits deamination of mc of cis-syn dimers at both TmC and mct sites flanked by adenines. The inhibition of deamination in the duplex may be due to the conformation of the flanking A s, which blocks the attack of water on the 5-methylcytosine group. For cis-syn dimers of a mct site, the rate of the deamination is 2.5 times slower in double-strand Ds than that in single-strand Ds, whereas the deamination rate of cis-syn dimers at TmC sites is about 6-fold slower in double-stranded Ds than in single-strand Ds. Replacing flanking A s by G s increases the rate of deamination at both mct and TmC sites in single-strand and double-strand Ds. This effect may originate from the ability of G to assist the addition of water to the 5-mC (slightly enhanced for 5 -mc and diminished for the 3 -mc).

20 By using tandem mass spectrometry coupled with an enzyme predigestion, we were able to study the deamination kinetics of 5-methylcytosine-containing CPDs. When flanked by adenines, the rate of deamination is faster in duplex DA. The rate is also faster at an mct site than at a TmC site in single-strand DA, whereas the rate is faster at TmC site than at mct site in duplex DA. When flanked by guanines, the rate increases for both single-strand and double-strand Ds compared to Ds with flanking by adenines. Deamination kinetics of CPDs in-vivo will be assessed to test whether these rate effects carry over to living systems.

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22 This research was supported by IH CA40463 (J.S.T.) and by the ational Centers for Research Resources of the IH, which supports the research resource at Washington University (Grant P41RR00954).