Study of Deamination Kinetics of 5-Methyl Cytosine-Containing Pyrimidine Dimers in

Oligodeoxynucleotides by Nuclease P1 Enzyme Digestion Coupled with Tandem

Mass Spectrometry

Bich T. N. Vu1, Yinsheng Wang2, Vairamani Mariappanadar3, John-Stephen A. Taylor1 and Michael L. Gross1

1) Washington University in St. Louis, Dept. of Chemistry, St. Louis, MO2) University of California at Riverside, Dept. of Chemistry, Riverside, CA

3) Indian Institute of Chemical Technology, Hyderabad, India

PurposeTo study the deamination kinetics of 5-methylcytosine in oligodeoxynucleotides (ODNs) containing cis-syn cyclobutanepyrimidine dimers (CPDs) photodamage. MethodsA quantitative nuclease P1 enzyme coupled with tandem mass spectrometry method was implemented to determine the deamination rate.ResultsThe deamination rates of 5-methylated cytosines in CPDswere obtained in single-strand and duplex ODNs, allowing us to begin to explore systematically the effects of sequence context, flanking sequence, and secondary structure on the rates of this important reaction.

The majority of mutations that cause skin cancer are C � T transitions or CC � TT mutations at dipyrimidine sequences resulting from UV-light-induced cyclobutane pyrimidine dimer (CPD) formation.

Methylation of cytosine enhances by 5-15-fold pyrimidine-dimerformation, resulting from exposure of cells to sunlight. The enhancement may be due to the higher UV absorptivity of 5-methylcytosine vs cytosine in DNA.

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,32P postlabeling was employed to quantify DNA photoproduct formation. We wish to continue to implement and apply Nuclease P1 enzyme-coupled ESI MS/MS as a quantitative method to study the deamination kinetics of 5-methylated cytosines in CPDs.

Photoproducts of DNA at Dipyrimidineand TA Sites

N

NO

5'

NH2

NH

N

3'

O

O

CH3 CH3

H H

N

NO

5'

NH2

NH

N

3'

O

O

CH3 CH3

H H

N

NO

5'

NH2

NH

N

3'

O

O

CH3 CH3

H H

[cis,syn] [trans,syn_I] [trans,syn_II]UVB (280-320nm) UVB

N

NO

5'

NH2CH3

H

OH

N

N

O

3'H3C

N

NO

5'

NH2CH3

H

OH

N

N

H3C 3'H

O

[Dewar][6,4]

UVB

UVB/A(280-400nm)

5’— TA NNNNN mCT — 3’UVB/C (240-320nm)

HN

NO

5'

OCH3

H

N

NH2

N

N N3'

TA* or T[ ]A

Deamination of 5-methylated Cytosine in UV-irradiated DNA

N

NO

5'

NH2

NH

N

3'

O

O

CH3 CH3

H H

HN

NO

5'

O

NH

N

3'

O

O

CH3 CH3

H H

H2O

5’—T[c,s]T —3’5’—mC[c,s]T —3’

HN

NO

5'

O

N

N

3'

NH2

O

CH3 CH3

H H

HN

NO

5'

O

NH

N

3'

O

O

CH3 CH3

H H

H2O

5’—T[c,s] T —3’5’—T[c,s]mC —3’

C � T and CC � TT Mutation Processes

5’ — GA — 3’3’— mCT — 5’

UVB

5’ — GA — 3’3’ — mC�T — 5’Error-prone

replicationDeamination

5’ — GA — 3’3’ — T�T — 5’

5’ — AA — 3’3’ —T�T— 5’

DNA pol. �

Replication

5’ — AA — 3’3’ — mC�T — 5’

Replication5’ — AA — 3’3’ — TT — 5’

• Nuclease P1 enzyme from Penicillium citrinum has been used to degrade DNA. It hydrolyzes single-strand DNA to 5’-mononucleotide, and photodamaged DNA to trinucleotides.

• For undamaged DNA and ODNs, the base � stacks with phenyl group in the active site of the enzyme, and the hydrolysis proceeds. The photodamaged site of DNA 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.

The Mechanism of Nuclease P1 Cleavage

Nuclease P1 enzyme cleaves undamaged DNA to mononucleotide and damaged DNA to trinucleotides.

d(GT[ ]ATTAT)

Phe

basedG, d(pT[ ]AT),d(pT), d(pA)

O H

O

OO PO

O

O

O

O

Enz

MaterialsODNs were obtained from IDT (Integrated DNA Technologies). Duplex hairpins were annealed by heating 1-mM solutions of ODNs to 90 �C prior to use.

Sample PreparationsSingle-strand and double-stranded ODNs containing cis-syn dimers were prepared by irradiating the starting ODN with 450-W mercury arc light �� ~300nm after filtration).The solutions containing cis-syn dimers of ODN 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.

AnalysisThe 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.

Nuclease P1 Enzyme Coupled Tandem Mass Spectrometry Assay

Identifying and quantifying photoproduct formationsDeamination— NT�mCN — — NT�TN —

P1 digestion

dpT�mCN dpT�TNdpN, dpN,

ESI coupled MS/MS

ESI coupled MS/MS

dpT�mC dpT�T

P1 digestion

m/z 803m/z 802

m/z

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.

• The five photoproducts, —N[cys,syn]NN—, —N[trans,syn_ I]NN—, —N[trans,syn_II]NN—, —N[6,4]NN—, —N[Dewar]NN—, after digestion with nuclease P1, give common products pN[ ]NN 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.

MS/MS of ESI-produced [M - H]- ions of Nuclease P1 Digestion Product for the cis-syn CPD

Characteristic fragments of cis-syn dimersDeamination

CACGTGmC[c,s]TGGCACCAC� Nuclease P1 digestion

d(pmC[c,s]TG) [M – H]¯ m/z 953

CACGTGT[c,s]TGGCACCAC� Nuclease P1 digestion

d(pT [c,s]TG) [M – H]¯ m/z 954

600 700 800 900

803

705

607 687 785Rel

ativ

e A

bund

ance

[M – G]

[M – G – H2O – HPO3]

[pT[c,s]pT – H2O]

[M – H]¯

1000m/z

MS/MS of ESI-produced [M - H]- ions of Nuclease P1 Digestion Product for the trans-syn CPD

Characteristic fragments of trans-syn dimers

d(GAGTAT[t,s_I]TATGAG)Nuclease P1digestion

d(pT[t,s_I]TA) [M – H]¯ m/z 938 938840

803

745705

400 600 800 1000

607Rel

. Abu

nd.

m/z

[M – H]¯ m/z 938

d(GAGTAT[t,s_II]TATGAG)Nuclease P1digestion

d(pT[t,s_II]TA) [M – H]¯ m/z 938

400 600 800 1000

803

745785705607R

el. A

bund

.

938

m/z

[M – H]¯ m/z 938

MS/MS of ESI-produced [M - H]- ions of Nuclease P1 Digestion Product for the 6-4 and Dewar Photoproducts

Characteristic fragments of 6-4 and Dewar dimers

Rel

. Abu

nd.

Rel

. Abu

nd.

960

847

825727432 749

400 600 800 1000

400 600 800 1000

960

847

825727432

m/z

m/z

d(GTAT[Dewar]TAT)[M + Na – 2H]¯ m/z 960

[M + Na – 2H]¯ m/z 960

Nuclease P1 digestion

d(T[Dewar]TA)[M – H]¯ m/z 938

d(GTAT[6-4]TAT)Nuclease P1 digestion

d(pT[6-4]TA)[M – H]¯ m/z 938

MS/MS of ESI-produced [M - H]- ions of Nuclease P1 Digestion Product for the TA Photoproduct

Characteristic fragments of TA photoproductGAGT[ ]ATmCATGAG

� Nuclease P1 digestiond(pT[ ]AT) [M – H]¯ m/z 938

600 m/z

616

803705607

500

Rel

ativ

e Ab

unda

nce

[M – H]¯ m/z 938[pTpA – H2O]

700 800 900 1000

MS/MS of ion of m/z 803, viewed in “Zoom-Scan” at Various Time Points of Deamination

m/z801.0 802.0 803.0 804.0 805.0

Rel

ativ

e Ab

unda

nce

0 min

120 min

240 min

720min

1800 min

802

803

804

�dpT[c,s]T – H�¯

� dpmC[c,s]T- H�¯

The peak of m/z 803 corresponding to deaminated component increases when deamination time increases, while the peak of m/z802 corresponding to undeaminated component decreases.

Evaluation of the 1st Order Rate Constant from MS Data

y = -0.00131x - 0.06727R2 = 0.99640

-2.5

-1.5

-0.5

0.5

0 500 1000 1500

y = -0.00199x + 0.06417R2 = 0.97200

-1.5

-1

-0.5

0

0.5

0 200 400 600 800Time (min)

ln(%

Und

eam

inat

ed)

GTGmC[c,s]TGGCACk = 0.00131 min-1

Hairpin CACGTGmC[c,s]TGGCACCACk = 0.00199 min-1

The fractions of deaminatedand 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.

Kinetic Parameters for 5-Methylcytosine Deamination in Cyclobutane Pyrimidine Dimers

650170390320

0.00106 � 15% 0.00416 � 4%

0.00178 � 12%0.00218 � 18%

GTGmC[c,s]TGGCACGTGT[c,s]mCGGCACHairpin CACGTGmC[c,s]TGGCACCACHairpin GTGT[c,s]mCGGCACCAC

Flanking Guanines

750420

17602600

0.000925 � 22%0.00165

0.00076 � 19%0.000266 � 23%

GAGTAmC[c,s]TATGAGGAGTAT[c,s]mCATGAGHairpin GCGCGTAmC[c,s]TATCGCGHairpin GCGCGTAT[c,s]mCATCGCG

Flanking Adenines

t1/2 (min)k (min-1)

37�C

Duplex formation inhibits deamination of mC of cis-syn dimers at both TmC and mCT sites flanked by adenines. The inhibition of deaminationin 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-syndimers of a mCT site, the rate of the deamination is 2.5 times slower in double-strand ODNs than that in single-strand ODNs, whereas the deamination rate of cis-syn dimers at TmC sites is about 6-fold slower in double-stranded ODNs than in single-strand ODNs.

Replacing flanking A’s by G’s increases the rate of deamination at both mCT and TmC sites in single-strand and double-strand ODNs. 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).

• By using tandem mass spectrometry coupled with an enzyme predigestion, we were able to study the deaminationkinetics of 5-methylcytosine-containing CPDs.• When flanked by adenines, the rate of deamination is faster in duplex DNA. The rate is also faster at an mCT site than at a TmC site in single-strand DNA, whereas the rate is faster at TmC site than at mCT site in duplex DNA.• When flanked by guanines, the rate increases for both single-strand and double-strand ODNs compared to ODNswith 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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• This research was supported by NIH CA40463 (J.S.T.) and by the National Centers for Research Resources of the NIH, which supports the research resource at Washington University (Grant P41RR00954).