In the late 1970's, Kohn and colleagues used the biophysical technique of alkaline elution to detect nitrogen mustard-induced DPCs in cells that had been exposed to mechlorethamine and melphalan (10
). However, no information was available regarding the identities of the proteins involved in DPC formation, nor were the chemical structures of the cross-linked lesions determined. Although previous work in our laboratory showed that mechlorethamine and chlorambucil cross-link the human DNA repair protein AGT to DNA in vitro
), questions remained about the ability of nitrogen mustard drugs to cross-link other nuclear proteins to DNA.
In the present work, mechlorethamine-induced DPCs formed in nuclear protein extracts from mammalian cells were isolated by affinity capture, followed by protein identification by mass spectrometry-based proteomics and western blotting. Analysis of DPCs isolated from mechlorethamine-treated nuclear protein extracts from CHO cells resulted in identification of 15 cross-linked proteins, while analogous experiments conducted using extracts from human cervical carcinoma (HeLa) cells resulted in positive identification of 53 proteins ( and , respectively). A considerable overlap was observed between the two protein lists. Two thirds of the CHO proteins that were identified following affinity capture of mechlorethamine-induced DPCs have human homologs that were detected in similar analyses of protein extracts from HeLa cells. The fact that a smaller number of proteins were identified in extracts from hamster cells is not due to more efficient DPC formation with human proteins, but is likely a result of an ascertainment bias resulting from the greater coverage of the human proteome (compared to the hamster proteome) in the databases used for protein identification (~135,000 versus ~1,600 proteins).
Among the proteins participating in DPC formation in the presence of mechlorethamine are those playing a role in cell motility (actin) (31
), transcriptional regulation (elongation factor 1-alpha 1, elongation factor 2, Ref-1, GAPDH), chromatin remodeling (actin, nucleolin) (32
), DNA supelcoiling (topoisomerases I and II) (35
), DNA replication (actin) (32
), glycolysis, initiation of apoptosis, and vesicle shuttling (33
) (GAPDH), ribosome biogenesis (nucleolin) (40
), and DNA repair (nucleolin, AGT, Ku70/86, XRCC1, XRCC5, PARP, Ref-1, Flap endonuclease 1) (13
). Some of these (e.g. actin, tubulin, nucleolin, HSP 90, and heterogeneous nuclear ribonucleoproteins) are high-abundance proteins (50
) that have a greater probability of cross-linking to DNA in the presence of bis
-electrophiles due to simple mass action considerations and, once affinity captured, are more likely to be identified by mass spectrometry. However, a subset of proteins identified upon analysis of mechlorethamine-induced DPCs (e.g. AGT, Ref-1, and Flap endonuclease 1) are present within the nucleus at much lower levels (50
). The latter proteins are involved in DNA replication and repair and probably have a higher propensity to form DPCs because of their strong affinity for DNA and their DNA binding mode that can bring monoalkylated nucleobases in a close proximity to active site residues (e.g. nucleotide flipping for AGT) (53
The distribution of proteins between functional classes was similar for proteins cross-linked to DNA in the presence of mechlorethamine and the total nuclear proteins found in MS analysis of the human nuclear proteome (54
), with the exception of proteins involved in DNA replication and repair which were over-represented among DPC-forming proteins (26% versus 10%). This suggests that proteins of this class are more susceptible to mechlorethamine-mediated DPC formation.
Recently, Qiu and Wang reported the use of formaldehyde cross-linking and tandem mass spectrometry to investigate DNA-protein interactions in vivo
). Following exposure of human acute promyelocytic leukemia cells to formaldehyde, proteins that had become cross-linked to chromosomal DNA were isolated, digested to peptides, and subjected to LC-MS/MS analysis. Identified proteins were then classified according to both cellular localization and function. Of the 780 proteins identified, 305 were classified as nuclear (~39%), and 46 were designated as DNA-binding proteins involved in DNA replication and repair processes (~6 % of all identified proteins) (55
). While there are definite similarities in terms of proteins identified as becoming cross-linked to DNA in the presence of both formaldehyde and mechlorethamine (including PARP, Ref-1, and Ku), the observed differences may be the result of varied reactivity/selectivity of the two agents. While formaldehyde is capable of reacting with the amino groups of guanine, adenine, and cytosine in DNA and lysine and arginine in proteins to form DPC lesions (56
), alkylation of biomolecules by nitrogen mustards is more selective, reacting primarily with the N7 position of guanine bases and the side chain sulfhydryls of cysteine residues (12
). The greater selectivity of nitrogen mustards in forming DPCs could account for the fewer cross-linked proteins identified following mechlorethamine exposure (14 versus 46 with formaldehyde), as well as the identification of a greater percentage of proteins specifically involved in DNA replication and repair (26% versus 6%).
An insight into the chemical structure of mechlorethamine-induced DPCs was provided by HPLC-ESI-MS/MS analyses of total proteolytic digests (). These results demonstrate that mechlorethamine-mediated DPCs are covalent in nature, involving the N7 position of guanine in duplex DNA and the side chain sulfhydryl of cysteine residues within nuclear proteins (N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine). The majority of the identified proteins contain at least one cysteine residue ( and ), suggesting that a similar amino specificity may be observed for other protein targets. While we did not observe the formation of the corresponding lysine-guanine cross-links via tandem mass spectrometry, cross-linking of other amino acids to DNA by mechlorethamine cannot be ruled out. Studies are currently underway which will allow for direct sequencing of modified peptides to identify the cross-linking sites.
Although the biological implications of DNA-protein cross-linking by mechlorethamine and other bis
-electrophiles remain to be determined, DPCs are bulky lesions that are expected to lead to genotoxic and cytotoxic effects if left unrepaired. Several types of cellular DPCs have been shown to be relatively long-lived, persisting through several rounds of DNA replication (6
). Thus, DPC formation could result in permanent DNA alterations and other negative consequences, if left unchecked. On the other hand, studies have shown that the majority of DPCs induced by exogenous agents are removed from the genome with time (6
), suggesting that these lesions are subject to cellular DNA repair. The mechanism(s) by which this occurs is currently under debate. Based upon the collective experimental evidence (59
), a model for the repair of covalent DPCs was proposed by Sancar and colleagues (61
). In this model, DPC repair is coupled to the replication-dependent proteolysis of cross-linked proteins. Once a DPC is encountered, a component of the stalled replication machinery sends a signal for proteolysis of the covalently attached protein to a peptide, followed by recruitment of proteins involved in nucleotide excision repair (NER). The resulting DNA-peptide cross-link is then removed via
nucleotide excision repair mechanism. More recently, Nakano et al.
examined the roles of NER and homologous recombination (HR) in the repair of structurally defined oxidative DPCs in vitro
and in bacterial cells (62
). While NER was involved in the removal of cross-linked proteins of relatively low molecular weights (<14 kDa), proteolytic degradation of DPCs did not contribute to DPC repair as Escherichia coli
cells deficient in cytosolic ATP-dependent proteases (counterparts of eukaryotic proteasomes) displayed similar results in terms of cell survival. Instead of NER-coupled proteolysis, HR repair was responsible for the removal of DPCs involving oversized proteins (>14 kDa) (62
Our study demonstrates that numerous nuclear proteins encompassing a variety of cellular functions can become covalently cross-linked to DNA in the presence of the representative nitrogen mustard, mechlorethamine. Although, for practical reasons, we employed much higher nitrogen mustard concentrations than are encountered clinically, it is reasonable to hypothesize that DPCs also form within the cells of individuals treated with nitrogen mustard drugs. Once formed, these DPCs could contribute to the cytotoxicity associated with these agents. For example, unrepaired DPCs could potentially catastrophically interfere with cellular transcription and/or replication, thereby triggering programmed cell death. On the other hand, mis-repair of these lesions could generate lethal mutations or result in chromosomal double-strand breaks, thereby activating programmed cell death. As an initial step towards testing these hypotheses, we are developing new strategies to selectively induce DPCs in a human cell culture model system.