Mammalian nucleotide excision repair proteins and interstrand crosslink repair

Abstract

Although various schemes for interstrand crosslink (ICL) repair incorporate DNA recombination, replication, and double-strand break intermediate steps, action of the nucleotide excision repair (NER) system or some variation of it is a common feature of most models. In the bacterium Escherichia coli, the NER enzyme UvrABC can incise on either side of an ICL to unhook the crosslink, and can proceed via a subsequent recombination step. The relevance of NER to ICL repair in mammalian cells has been challenged. Of all NER mutants, it is clear that ERCC1 and XPF-defective cells show the most pronounced sensitivities to ICL-inducing agents, and defects in ICL repair. However, there is good evidence that cells defective in NER proteins including XPA and XPG are also more sensitive than normal to ICL-inducing agents. These results are summarized here, together with evidence for defective crosslink removal in NER-defective cells. Studies of incision at sites of ICL by cell extracts and purified proteins have been done, but these studies are not all consistent with one another and further research is required.

Figure 1

Two of the possible roles for NER in processing of ICL. A, dual incision on either side of a crosslink; B, dual incision to remove a crosslink unhooked on one strand, for example after translesion DNA synthesis has re-synthesized DNA on an initially processed strand.

Figure 2

Diagram of events leading to dual incision in the human NER system for an adduct on one strand, such as a UVC-induced (6-4) photoproduct. The proteins are shown roughly to scale, relative to DNA. In step (i), XPC-RAD23B binds preferentially to a site of DNA where base pairing is disrupted by lesion. At least 17 bp of DNA are contacted, including the nondamaged DNA strand opposite the lesion (Min and Pavletich 2007). XPC-RAD23B binds TFIIH, which enters the DNA with other components in step (ii), where a pre-incision complex is formed including TFIIH of dimensions ∼ 16 nm × 12.5 nm × 7.5 nm, (Schultz et al. 2000), XPA, RPA (which includes 4 active DNA-binding OB fold domains amongst its 3 subunits), and XPG. The DNA strands are separated in this step in an ATP-dependent reaction (Evans et al. 1997a). In step (iii), the XPF-ERCC1 nuclease complex produces a 5′ incision and the XPG nuclease the 3′ incision. The two incisions take place nearly simultaneously after formation of a productive pre-incision complex. The CDK7-activating (CAK) subunits of TFIIH are not present in the fully formed pre-incision complex and detach from core TFIIH (Coin et al. 2008). In step (iv), after dual incision, a DNA polymerase holoenzyme utilizing the sliding clamp PCNA fills in the gap created by incision, and a single-stranded DNA piece containing the lesion is released.