Because DNA ligases as well as gap-filling DNA polymerases require 3′-OH substrates, removal of 3′-PG termini is an essential early step in the repair of free radical-mediated DSBs. Candidates for carrying out such removal include dedicated 3′-terminal processing enzymes such as Ape1, PNKP and TDP1, as well as 3′ exonucleases such as DNase III, Mre11 and Wrn. However, based on the known specificities of these enzymes, none of them except TDP1 is expected to act on protruding 3′-PG termini of DSBs. Ape1 acts only on blunt and recessed 3′ ends (8
), and neither PNKP nor DNase III has any activity toward 3′-PG termini (11
). Although the activities of Mre11 and Wrn toward modified termini have not been rigorously examined, they both show a strong preference for recessed 3′ termini, and 3′ overhangs are extremely poor substrates for these enzymes even when they bear normal 3′-OH termini (33
). The Saccharomyces cerevisiae
Rad1/Rad10 endonuclease (and presumably its human ERCC1/XPF homolog) can remove a single 3′-PG (or 3′-OH) nucleotide from the 3′ end of a single-strand break in double-stranded DNA (36
), but no such activity has been reported on PG-terminated 3′ overhangs. Similarly, while Artemis, a DNA-PK-activated endo/exonuclease, has been implicated in DSB repair (37
), this enzyme appears to act only on 3′ overhangs longer than four bases (38
Consistent with these predictions, in extracts of SCAN1 cells harboring mutant TDP1, there was no detectable processing of a PG terminus on a three-base 3′ overhang. Moreover, despite the fact that purified TDP1, whether produced in mammalian (data not shown) or bacterial (11
) cells, is ~100-fold less active toward 3′-PG than toward 3′-phosphotyrosyl substrates, the levels of TDP1 and PNKP in both whole-cell and nuclear extracts of normal cells are sufficient to rapidly convert any exposed 3′-PG termini to 3′-phosphate and then to 3′-OH termini (although in nuclear extracts DSB ends appear to be partially shielded from such processing). Both these results are consistent with the view that most if not all processing of protruding 3′-PG termini is dependent on TDP1.
Because a substantial proportion, perhaps as many as half, of DNA breaks induced by ionizing radiation bear 3′-PG termini (7
), cells deficient in processing of PG-terminated DSBs are expected to be profoundly radiosensitive. However, in cell growth assays, only slight radiosensitivity was detected in two of three SCAN1 cell lines, and then only for fractionated radiation in plateau phase. These results suggest that most PG-terminated DSBs are still repaired in SCAN1 cells, presumably by TDP1-independent pathway(s). As noted above, blunt and recessed 3′-PG ends could be processed by Ape1, while 3′ overhangs longer than four bases could be processed by Artemis. Moreover, for DSBs with one 3′-PG and one 3′-phosphate terminus, it is possible that the strand with the 3′-phosphate could first be rejoined, and then the 3′-PG strand could be processed as if it were a single-strand break, with PG removal by Ape1. Thus, the fraction of radiation-induced DSBs with a strict requirement for TDP1 (perhaps only those with one- to four-base PG-terminated 3′ overhangs in both strands) may be quite small, possibly <10%. Finally, even for these breaks, there may be alternative, more complicated repair pathways that can substitute when simpler end-joining pathways fail, as suggested by the ‘repair foci’ of DSB repair factors that can be detected cytologically long after the majority of DSBs have already been repaired (39
). Failure of these complexes to form properly in cell extracts could explain the complete lack of apparent processing in extract-based assays. Whether protruding PG termini on DSBs are more persistent in SCAN1 than in normal cells in vivo
is not known, and although a post-labeling assay for PG formation and repair in vivo
has been described previously (41
), this assay is relatively insensitive and does not distinguish between single- and double-strand break termini. Thus, further work will be required to resolve the apparent disparity between the profound repair deficiency seen in cell extracts and the relatively mild radiosensitivity of SCAN1 cells.
Similar to many DNA repair proteins (28
), TDP1 is phosphorylated, and its phosphorylation is stimulated by ionizing radiation, providing indirect evidence of possible involvement in pathways for repair of radiation-induced damage. The kinase(s) involved have yet to be identified, but phosphorylation on serine/threonine residues as well as stimulation by ionizing radiation would be consistent with ATM and/or DNA-PK (28
). Phosphorylation is clearly not required for the basic enzymatic function of TDP1, as recombinant proteins produced in bacteria or in human cells show qualitatively similar activity (H. Tatavarthi and L. F. Povirk, unpublished data). However, phosphorylation could modulate TDP1 activity or influence its interactions with other repair proteins.
Although not proven, it is generally assumed that SCAN1 pathology is a consequence of the failure of mutant TDP1 to efficiently repair topoisomerase I-associated DNA damage (12
). This repair deficiency could indirectly confer sensitivity to oxidative DNA damage, as certain oxidative lesions tend to promote formation of topoisomerase I cleavable complexes (46
), which upon replication can be converted to cytotoxic topoisomerase-terminated DSBs (47
). Nevertheless, a role for PG-terminated DSBs in SCAN1 pathology cannot be excluded. The neuronal apoptosis seen in knockout mice lacking critical DNA end-joining proteins, such as XRCC4 and DNA ligase IV (48
), suggest that in the absence of DSB repair, terminally differentiating neurons accumulate sufficient DSBs to produce significant neurological pathology. There is evidence that at least some of these ‘spontaneous’ DSBs reflect damage by oxygen free radicals associated with normal oxidative metabolism (50
). Moreover, SCAN1 pathology is similar to that of both Friedreich ataxia and ataxia with oculomotor apraxia (AOA1) (51
), both of which have been linked to oxidative damage. Friedreich ataxia is associated with a mutant frataxin protein, which leads to increased oxidative stress due to a defect in iron homeostasis (52
). AOA1 cells are sensitive to hydrogen peroxide and harbor a mutation in aprataxin; while the exact function of the aprataxin protein is not known, it interacts with the single-strand break repair protein XRCC1. In S.cerevisiae
of appropriate genetic backgrounds, TDP1 deficiency confers sensitivity to ionizing radiation as well as to bleomycin, a radiomimetic drug that specifically induces PG-terminated DSBs (57
). Thus, while the exact sequence of events culminating in the SCAN1 phenotype remains to be elucidated, several lines of circumstantial evidence suggest a possible linkage among TDP1 deficiency, oxidative damage, DSBs and cerebellar ataxia. Even though lymphoblastoid cell lines derived from SCAN1 patients show at most only mild radiosensitivity, it is possible that endogenous tissues of SCAN1 patients are more sensitive to free radical damage than the cell lines. Because cultured cells are subjected to higher oxygen tensions than would occur in vivo
, they may be more likely to adapt to TDP1 deficiency, for example, by upregulating alternative pathways for repair of DSBs and other oxidative damage.