Covalent interstrand DNA cross links (ICLs) between complementary stands of the DNA double helix constitute a major and particularly cytotoxic class of DNA damage (1). As these crosslinks affect both DNA strands, the redundant information normally safeguarded by the DNA double helix is jeopardized, and cellular repair processes can induce double strand breaks and other potentially deleterious lesions in the process of removing the lesion (2, 3, 4). These highly toxic ICLs primarily disrupt rapidly dividing cells as they pose physical obstacles to strand separation at the replication fork and to transcriptional bubble formation. Consequently, structurally diverse agents such as psoralen, mitomycin C, cisplatin, and nitrogen mustards that induce ICLs have become important chemotherapeutics and frontline weapons against many types of cancer (1, 2, 3). ICL repair in mammalian cells proceeds via several overlapping pathways, and the precise repair mechanism depends upon the nature of the crosslink, and the stage of the cell cycle when the damage is discovered (2, 5, 6). Prominent among these pathways is replication-dependent repair (S phase), where the ICL poses a physical barrier to the progressing replication fork, and stalled replication complexes lead to the recruitment of repair machinery that initiates repair via ICL-induced double-strand breaks (5). Similarly, ICLs encountered at or near actively transcribed regions of the genome during Go or G1 stages of the cell cycle can prevent transcriptional bubble formation and stall transcriptional machinery (2, 3, 7). Such transcription-coupled repair (TCR) processes involve recruitment of nucleotide excision repair (NER) factors to the stalled complex (8, 9, 10, 11). Finally, ICL detection in Go or G1 can also occur in unperturbed DNA in a process known as the global genome repair (GGR) pathway of NER, which occurs in the absence of transcription (7, 10, 11, 12). The GGR pathway has only been completely worked out in prokaryotes, and involves NER-dependent incision events on a single DNA strand on both the 5′ and 3′ sides of the ICL resulting in “unhooking” of the crosslink. However, previous studies using mammalian cell extracts showed that NER only leads to dual incisions on the 5′ side of the ICL that do not result in crosslink unhooking (Figure 1) (13). Figure 1 Schematic illustrating NER-dependent and -independent processing of ICLs observed in mammalian cell extracts (32). Interstrand crosslinks prevent mammalian NER enzymes from making incisions surrounding the crosslink. Instead, unknown repair enzyme activities ... Recently there has been increased interest in the process of ICL detection using model ICL-containing duplexes that serve as defined substrates for DNA repair factors present in mammalian cells(Fig. 1) (11, 14, 15). A model ICL that has been very useful in ex vivo repair studies is one in which the exocyclic N4 nitrogen atoms of staggered cytosine bases on opposite strands of the DNA duplex are connected by an ethyl covalent linker (Fig. 2). This defined crosslink can be site-specifically incorporated using a convertible nucleoside approach and has been extensively characterized using structural and biochemical methodology (2, 16, 17, 18). Due to the inherent anti-parallel nature of the DNA duplex, two different orientations of the staggered CC ICL can be generated, one in which the crosslink is placed in a 5′-CG-3′ sequence and one in which the crosslink is placed in a 5′-GC-3′ sequence. Because of the relative geometries of the staggered cytosine bases in these two forms, only the 5′-CG-3′ orientation allows an ethyl linker geometry that optimally positions the N4 atoms of the linked cytosine bases to maintain normal Watson-Crick hydrogen bonding contacts with their cross strand partner guanine, thereby maintaining the B-form DNA structure (Fig. 2b). 5′-CG-3′ ICL-DNA has been structurally characterized in different sequence contexts using x-ray crystallography, solution state NMR spectroscopy, and atomic force microscopy, and found to adopt a structure that is essentially identical to canonical B-form DNA (16, 18, 19). In contrast, the non-optimal 5′-GC-3′ ICL-DNA form has been found to have a highly distorted duplex structure (16). Figure 2 Structure of DNA duplex containing the N4C-ethyl-N4C crosslink. (a) Crystallographic model of duplex DNA (pdb accession name 2OKS) containing a central staggered 5′–CG-3′ N4C-ethyl-N4C interstrand crosslink in the major groove. ... When these staggered CC ICL duplexes were exposed to mammalian cell extracts it was observed that the disordered 5′-GC-3′ construct and the B form 5′-CG-3′ ICL-DNA were incised in two distinct ways (14, 20), but a matched normal DNA was not incised. The first set of incisions occurred on a single strand, 5′ to the ICL and produced a set of oligonucleotides 24–32 nucleotides in length (Fig. 1), similar to those previously reported by Sancar and coworkers (13). The second set of apparent incisions appeared on both the 5′ and 3′ sides of the ICL and led to unhooking of the ICL (Fig. 1) 2. Through the use of extracts derived from cells that were deficient in NER enzymes, it was established that the dual 5′ incisions require NER, while the highly-specific 5′ and 3′ incision activity that leads to unhooking is novel, and not attributable to known DNA repair pathways (14). The disordered 5′-GC-3′ ICL was unhooked with an initial rate that was 6-fold grater than that of the B-form 5′-CG-3′ ICL-DNA, suggesting that DNA distortion plays a role in detection, however the remarkable aspect of both NER-dependent and independent incision events is that the normal matched DNA duplex is never detectably incised (14). Thus, subtle differences in the structure and/or dynamic properties of the B form ICL and the normal duplex must give rise to incision specificity. While the gross structural distortions provided by the 5′-GC-3′ ICL could lead to its preferred incision (14, 16), the detection of specific incision events in the structurally undistorted 5′-CG-3′ ICL duplex demonstrates that structural distortions can not be the sole determining factor guiding incision. Since structural methods only yield information on the time-averaged conformation of a macromolecule, they are insensitive to short-lived high-energy states that may be present and critical to biological recognition (21, 22, 23, 24). Here we use NMR imino proton exchange measurements to probe the dynamics of B-form and non-B-form duplexes containing staggered CC ICLs. We find that the intrinsic dynamic properties of these ICL-DNAs do not provide an adequate explanation for their specific endonucleolytic incision by DNA repair factors in mammalian cell extracts. The findings suggest that the covalent nature of the two linked DNA strands is the essential determining feature for incision of both ICL’s, and that the differences in the dynamic and structural properties of the two ICL duplexes plays a lesser role. A model for recognition and incision is proposed based on these findings.