Repair of Laser-localized DNA Interstrand Cross-links in G1 Phase Mammalian Cells

Abstract

Interstrand cross-links (ICLs) are absolute blocks to transcription and replication and can provoke genomic instability and cell death. Studies in bacteria define a two-stage repair scheme, the first involving recognition and incision on either side of the cross-link on one strand (unhooking), followed by recombinational repair or lesion bypass synthesis. The resultant monoadduct is removed in a second stage by nucleotide excision repair. In mammalian cells, there are multiple, but poorly defined, pathways, with much current attention on repair in S phase. However, many questions remain, including the efficiency of repair in the absence of replication, the factors involved in cross-link recognition, and the timing and demarcation of the first and second repair cycles. We have followed the repair of laser-localized lesions formed by psoralen (cross-links/ monoadducts) and angelicin (only monoadducts) in mammalian cells. Both were repaired in G 1 phase by nucleotide excision repair-dependent pathways. Removal of psoralen adducts was blocked in XPC-deficient cells but occurred with wild type kinetics in cells deficient in DDB2 protein (XPE). XPC protein was rapidly recruited to psoralen adducts. However, accumulation of DDB2 was slow and XPC-dependent. Inhibition of repair DNA synthesis did not interfere with DDB2 recruitment to angelicin but eliminated recruitment to psoralen. Our results demonstrate an efficient ICL repair pathway in G 1 phase cells dependent on XPC, with entry of DDB2 only after repair synthesis that completes the first repair cycle. DDB2 accumulation at sites of cross-link repair is a marker for the start of the second repair cycle. Interstrand cross-links (ICLs)2 are among the most dangerous DNA lesions. They are absolute blocks to replication and transcription and, unlike monoadducts, cannot be carried through a proliferative cycle. Their accumulation over time is believed to contribute to genomic instability and aging in tissues and organs (1, 2). If not removed, they can provoke chromosomal breakage, rearrangements, or cell death (3, 4). Mice and humans deficient in genes associated with ICL repair, such as members of the ERCC1-XPF complex, display severe pathology and greatly reduced life span (2, 5-9). Given the challenge of repairing lesions that engage both strands of the duplex, it is understandable that multiple repair pathways are engaged (10 -12) and that repair is more complex than for monoadducts.In Escherichia coli, the NER apparatus incises one strand on either side of the ICL. This produces an "unhooked" substrate with the excised fragment still attached to the non-incised strand by the cross-linking agent. The immediate product of unhooking is typically depicted as a gapped structure with the cross-linked oligonucleotide flipped out of the duplex (although this structure may not actually form (13)). The incised/gapped strand is repaired by homology-directed repair using information from an undamaged homologous chromosome (14 -17). The gap may also be filled by lesion bypass sy...

NEIL1 Responds and Binds to Psoralen-induced DNA Interstrand Crosslinks

“…Prior studies have shown that the NER damage recognition factor, XPC, which exists as part of a complex with HR23B, is an early arriver to DNA interstrand crosslink damage and directs a conventional repair response (16). To determine whether XPC facilitates the observed NEIL1 accumulation, we examined GFP-NEIL1 recruitment dynamics in cells deficient in XPC.…”

“…In these experiments, photoactivated trioxsalen (psoralen) is used as the interstrand crosslinking agent, and angelicin is used as a control to generate monoadducts exclusively (Ref. 16; further details below). Our studies herein focus on the NEIL1 DNA glycosylase, since this protein has been previously shown to play multiple roles in interstrand crosslink repair (10,12).…”

“…Psoralen Repair Kinetics-Control and NEIL1 knockdown cells (Trevigen) were seeded (2 ϫ 10 5 ) in a 35-mm glass bottom culture dish for 24 h. These cells were treated with 20 M digpso and incubated at 37°C for 30 min prior to laser treatment at 1.7% intensity (14,16). After the laser treatment, cells were fixed either immediately or at the indicated time point in 4% formaldehyde for 10 min at room temperature.…”

“…Treatments were carried out at the designated concentrations of trioxsalen (plus UVA light using a UVA Rayonet box from Southern New England Ultraviolet Company (Branford, CT)) as outlined previously (16). Cells were then gently washed twice with 1ϫ PBS, and incubated for 10 days with fresh DMEM to allow individual colonies to form.…”

“…A setting of 1.7% (for psoralen) and 2.2% (for angelicin) was used to create interstrand crosslinks or monoadducts in the targeted region. A setting of 2.7% was used to create free radical induced single strand breaks (16). 30 min prior to the experiment, cells (as indicated) were treated either with 5 M psoralen (30 min at 37°C), 40 M angelicin (30 min at 37°C), or not at all.…”