Repair of genomic interstrand crosslinks

Abstract

Genomic interstrand crosslinks (ICLs) are formed by reactive species generated during normal cellular metabolism, produced by the microbiome, and employed in cancer chemotherapy. While there are multiple options for replication dependent and independent ICL repair, the crucial step for each is unhooking one DNA strand from the other. Much of our insight into mechanisms of unhooking comes from powerful model systems based on plasmids with defined ICLs introduced into cells or cell free extracts. Here we describe the properties of exogenous and endogenous ICL forming compounds and provide an historical perspective on early work on ICL repair. We discuss the modes of unhooking elucidated in the model systems, the concordance or lack thereof in drug resistant tumors, and the evolving view of DNA adducts, including ICLs, formed by metabolic aldehydes.

Keywords: Cancer chemotherapy; DNA repair; Drug resistance; Interstrand crosslink; Unhooking.

Figure 1

CL forming compounds.

Figure 2.

Structure of DNA containing ICLs introduced by clinically important compounds. A. The distortion induced by psoralen, nitrogen mustard, and cisplatin (adapted from [312], PDB ID: 1A2E, 204D). B. While the colibactin ICL between adenines has been characterized the influence on DNA structure has not been determined.

Figure 3.

Early models of ICL repair. A. The Cole-Sinden model of replication independent repair. Incisions are introduced on either side of an ICL resulting in a gap with an attached crosslink remnant. The gap is filled either by a recombinational mechanism ([121] or by TLS [123, 124]. The crosslink remnant is then repaired by nucleotide excision repair. B. Unhooking by incision coupled to exonucleolytic digestion past the ICL [136]. C. Stalled Single Fork: A stalled single fork is the substrate for incisions on the leading template strand on either side of an ICL resulting in a single sided DSB on the leading strand sister chromatid and a gap with the crosslink remnant on the other sister chromatid. TLS past the crosslink remnant converts the lagging side sister chromatid to an intact duplex. Following excision repair of the crosslink remnant the fork is rebuilt by HR of the single sided DSB with the restored sister chromatid. This model required an unidentified activity to resume replication [140].

Figure 4.

Models of replication dependent repair pathways. A. Double Fork Convergence: Two forks coming from opposite directions collide with the ICL. After removal of the replisomes one of the forks reverses producing a duplex region appropriate for recruitment of, and incision by, XPF/ERCC1. Exonuclease digestion by SNM1A into the parental template strand of the leading daughter unhooks the ICL generating a leading strand sister chromatid with a double sided DSB while the other sister chromatid has a gap and the crosslink remnant. Restoration of the reversed fork enables TLS past the crosslink remnant, which would be removed by excision repair. The sister chromatid with the double sided DSB is then repaired by HR using the intact sister chromatid as the template. This yields two intact sister chromatids with no requirement for further DNA synthesis [8]. B. ICL Reversal by NEIL3: Fork convergence at ICLs formed by psoralen triggers cleavage by NEIL3 of the glycosidic bond between one of the crosslinked bases and the sugar yielding a crosslink remnant consisting of psoralen linked to one strand and a thymine residue. The other strand has an abasic site. With an ICL formed by an abasic aldehydic sugar cleavage by NEIL3 restores the adenine on the one strand leaving an abasic site on the other. No double strand breaks are formed, and crosslink remnants are bypassed by TLS. As implied by the dual fork convergence, DNA synthesis is completed prior to unhooking by NEIL3 [181].

Figure 5.

Replication/transcription independent repair. A distorting ICL is recognized by MutSα followed by incision and exonucleolytic digestion by MutLα and EXO1. Gap filling precedes removal of the crosslink remnant by excision repair [202].

Figure 6.

Replication traverse of genomic ICLs. A. Experimental scheme: antigen tagged psoralen ICLs are introduced into cells followed by incubation with CldU and then IdU. B. A replisome drives a replication fork. C. A single fork stalls at a tagged ICL. D. The stalled fork might be joined by a converging fork, or E. Replication traverse of the ICL with the fork moving in the same direction as before the encounter. F. An X structure forms which would enter the same repair sequence as described for the double fork collision model [231].