Abstract
BackgroundMechlorethamine [ClCH2CH2N(CH3)CH2CH2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown.Methodology/Principal FindingsHPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair) indicated formation of an interstrand crosslink at m/z 9222.088 [M−2H+Na]+. Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH2CH2N(CH3)CH2CH2] at m/z 269.2 [M]2+ (expected m/z 269.6, exact mass 539.27) and its hydrolytic product dC-mech-OH at m/z 329.6 [M]+ (expected m/z 329.2). Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M]+, which are both due to loss of the 4-amino group of cytosine (as ammonia), in addition to dC and dC+HN(CH3)CH = CH2, respectively. The presence of m/z 269.2 [M]2+ and loss of ammonia exclude crosslink formation at cytosine N4 or O2 and indicate crosslinking through cytosine N3 with formation of two quaternary ammonium ions.ConclusionsOur results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N3 position of cytosine.
Highlights
DNA damage and mutation can have major effects on genetic information that may alter the function of essential proteins and cause disease
Our results provide an important addition to the literature, as the first example of the use of High performance liquid chromatography (HPLC) and mass spectrometry (MS) for analysis of a DNA adduct at the N3 position of cytosine
We proposed that the mechlorethamine C-C crosslink forms in the DNA minor groove through the N3 of cytosine [19], but at the time we were unable to prove the atomic connectivity of the crosslink
Summary
DNA damage and mutation can have major effects on genetic information that may alter the function of essential proteins and cause disease. Sources of mismatch base pairs include replication errors due to direct misincorporation of bases, lesions in the parent strand, and formation of a heteroduplex between two homologous DNA molecules during recombination [2,3,4]. Mismatch base pairs cause thermodynamic instability of DNA duplexes [8], but most retain an intrahelical conformation with one or more hydrogen bonds between the bases: examples include A-A [9], G-G [9], A-C [10], G-T [11], C-T [12] and C-C [12,13,14] pairs. Establishment of DNA structures containing mismatch base pairs is important for understanding their involvement in replication, repair, and recombination. Mechlorethamine [ClCH2CH2N(CH3)CH2CH2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. The atomic connectivity of this unusual crosslink is unknown
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