Figure summarizes the findings of this study. Psoralen- and TBG-cross-linked linear DNA duplexes are specifically degraded by XPF-ERCC1 in the presence of RPA, producing two types of products. The first (Fig. A, products a and b) is the result of 3′-to-5′ exonucleolytic digestion, which is highly attenuated immediately past the cross-link site. The second types of products (products c and d) result from complete digestion of one strand by the 3′-to-5′ exonuclease activity by XPF-ERCC1. Given the exquisite sensitivity of rodent XPF and ERCC1 mutant cells to cross-linking agents, it is possible that the products in Fig. A are recombinogenic and potential substrates for XRCC2- and XRCC3-dependent steps of cross-link repair. Thus, it is conceivable that upon encountering a replication fork, the cross-link may give rise to a double-strand break (24
) which creates an entry site for XPF-ERCC1 to process the DNA into a form which is then acted upon by XRCC2 and XRCC3 to generate a cross-link-free duplex. Further work is needed to test such a model.
FIG. 10 Summary of the main findings of this study. (A) Cross-link-specific exonucleolytic degradation of a linear duplex by XPF-ERCC1 in the presence of RPA. Four types of products are generated. Products a and b result from the cross-link-attenuated progression (more ...)
Figure B summarizes the processing of psoralen cross-links by the mammalian nucleotide excision repair system which excises 22- to 28-nt oligomers from the 5′ side of the cross-link (1
). In this reaction, the pyrone-side adducted strand is preferred over the furan-side adducted strand by a factor of about 10 to 1. The structural and mechanistic basis of this preference is unknown, but it is of interest that it is the opposite of that of the E. coli
excinuclease, which prefers the furan-side adducted strand (32
) and which makes dual incisions bracketing the cross-linked bases. The resulting gap is filled by DNA polymerases in both cases. However, in mammalian cells the repair patch terminates at a nick adjacent to the cross-link in 90% of cases and is ligated to the parental DNA to regenerate the cross-linked substrate in the remaining 10% of molecules.
The reactions summarized in Fig. are dependent on the excision repair system but independent of the XRCC2 and XRCC3 proteins which have been found by genetic studies to be required for the major pathway of cross-link repair (35
). These results raise two interrelated issues: are the dual incisions 5′ to the cross-link and the subsequent futile DNA synthesis relevant to cross-link repair, and what are the likely roles of XRCC2 and XRCC3 in cross-link repair? We do not have the answers to these questions. However, both the dual incisions and the futile DNA synthesis are such efficient reactions in vitro that we are inclined to believe that they occur with reasonably high frequency in vivo and may play some important roles in the cellular response to DNA cross-links. Thus, the excised oligomer may be a coactivator for an SOS-like response in mammalian cells and the gap filling by DNA polymerase
may activate the DNA damage checkpoint response (31
). Furthermore, it is possible that the repair synthesis which generates a nick immediately 5′ to the cross-link will produce a prerecombinogenic structure which eventually leads to cross-link removal by recombination. However, we failed to see further processing of the nicked intermediate in either the presence or absence of XRCC3 protein, which is believed to be required for cross-link repair. XRCC2
encode RecA/HsRad51 homologs which are involved in homologous recombination (16
) and are thought to catalyze strand transfer and thus to participate in cross-link repair by promoting the recombination of the cross-linked duplex with a homologous duplex with no damage (35
). Hence, to provide a substrate for recombination, we included into our reaction mixtures homologous DNA duplex with no damage, single-stranded circular, or linear fragments complementary to the entire cross-linked plasmid or to the cross-linked region. Under no circumstances were we able to detect the elimination of cross-links from the substrate (data not shown).
In vivo data with cellular DNA (13
) or transfected plasmid DNA (7
) clearly show that mammalian cells are capable of removing interstrand cross-links. Indeed, previously, using randomly cross-linked DNA, we obtained data suggesting the removal of interstrand cross-links in vitro (30
). In that study, however, the conclusion that repair synthesis was accompanied by the disappearance of cross-links was based on indirect evidence. In light of the results obtained in the present work with a substrate containing a single cross-link, we conclude that most of the cross-link-induced DNA synthesis observed in the previous study was not associated with cross-link removal. The data herein also constitute unambiguous evidence for damage-induced DNA synthesis that is not repair synthesis in the strict sense of the word. Finally, the “repair synthesis” results presented in this paper differ from those in a recent report suggesting that cross-link-induced DNA synthesis was independent of nucleotide excision repair but dependent on XPF-ERCC1, XRCC2, and XRCC3 and was greatly stimulated by homologous or nonhomologous DNA (18
). As documented in Fig. , our repair synthesis (i) depends on a functional excision repair system, (ii) is independent of XRCC3, and (iii) is not affected by the presence of a second plasmid with or without homologous sequence in the reaction mixture (data not shown). We have no satisfactory explanations for the seemingly contradictory results of these two studies. However, in the previous study, cross-link-induced DNA synthesis was analyzed only in terms of radiolabel incorporated into the entire plasmid or large restriction enzyme fragments separated on nondenaturing agarose gels (18
). Hence, it is not possible to ascertain whether the cross-link-induced DNA synthesis in that study was confined to the area of the cross-link, whether the newly synthesized DNA was ligated to the parental DNA, and whether there was preferential DNA synthesis in the region of homology when homologous nondamaged DNA was included in the reaction mixture. Clearly, more studies are needed with well-defined substrates and purified enzymes to gain better insight into the mechanism of cross-link repair in mammalian cells.
Finally, we would like to comment on the potential clinical significance of the cross-link-induced futile DNA synthesis. The concept of futile repair is currently being discussed with respect to both mismatch repair activity on damaged DNA (25
) and transcription-coupled repair at transcription pause sites (11
), but with little direct evidence. The work presented in this paper is the only direct demonstration so far of a potentially futile repair cycle in mammalian cells. It is plausible that this futile cycle and the potentially prpapoptotic signals resulting from this cycle, rather than the replication block per se, are the main causes of the lethality of cross-link-inducing anticancer drugs.