A new photoproduct of 5-methylcytosine and adenine characterized by high-performance liquid chromatography and mass spectrometry

Abstract

The UV portion of sunlight is mutagenic and can modify DNA by producing various photoproducts. UV photodamage often occurs at dipyrimidine sites, to give cyclobutane, pyrimidine-(6-4)-pyrimidone (6-4), and pyrimidine-(6-4)-Dewar pyrimidone (Dewar) photoproducts, and at TA and AA sites. There is no reported evidence, however, of UV photoproduct formation between C or 5-methylC ((m)C) and A. Irradiation of d(GTAT(m)CATGAGGTGC) with UVB light at physiological pH gives an unexpected photoproduct that undergoes fast thermal deamination but does not revert to its original structure under UVC irradiation. Evidence from nuclease P1 digestion coupled with electrospray ionization (ESI)-MS/MS is in accord with product formation between (m)C and A. HPLC analysis indicates that deamination gives a T<>A photoproduct that coelutes on reverse-phase chromatography with the well-known TA* photoproduct, formed from an initial [2 + 2] reaction between C5-C6 and C6-C5 of the adjacent thymine and adenine [as shown by Zhao , X. , et al. ( 1996 ) Nucleic Acids Res. 24 , 1554 - 1560 and Davies , R. J. , et al. ( 2007 ) Nucleic Acids Res. 35 , 1048 - 1053 ]. Furthermore, the deamination product of the unknown (m)C<>A photoproduct and the TA* photoproduct undergo nearly identical fragmentation in tandem MS. The evidence, taken together, indicates that the deamination product of the unknown (m)CA photoproduct has the same chemical structure as the TA* photoproduct. Therefore, the unknown photoproduct is referred to as the (m)CA* photoproduct, which, upon deamination, gives the TA* photoproduct.

Figure 1

RP-HPLC of UVB-induced photoproducts of d(GTAT^mCATGAGGTGC) at pH 7.6 (upper panel) and of corresponding deamination products (lower panel) with A) 302 nm light and B) 312 nm light. SM: starting material, * indicates photoproduct that has undergone deamination

Figure 2

RP-HPLC assay of A) ~ 5 nmol UVC irradiation mixture of d(GTATTATGAGGTGC), B) ~ 360 pmol of deamination product of HPLC fraction of d(GTAT^mCATGAGGTGC), and C) a coinjected sample of A and B. # is the contamination peak of PP3 from HPLC separation (Figure 1A, lower panel)

Figure 3

NP1 coupled ESI- MS² and MS³ assays of mC<>A photoproduct of d(GTAT^mCATGAGGTGC) following deamination to T<>A (A and B, respectively), and TA* photoproduct of T5A6 of d(GTATTATGAGGTGC) (C and D, respectively).

Scheme 1

Known photoproducts that could originate from the designed sequence.

Scheme 2

Proposed structure of the ^mC<>A photoproduct and its mechanism of formation and deamination.