Cernunnos was discovered as the gene defective in a subset of SCID patients with accompanying microcephaly (2
), and independently as XLF, a binding partner of XRCC4 (1
). It has a structure similar to that of XRCC4, with a globular head and long α-helical tail (3
). Cernunnos binds to Ku as well as to XRCC4, and induces supershifts of both Ku-DNA and XRCC4-DNA complexes in electrophoretic mobility shift assays (3
). The cellular phenotype for Cernunnos deficiency is similar to that for Ku or XRCC4, and includes radiosensitivity and failure to rejoin a substantial fraction of radiation-induced DSBs (2
). In reactions with purified proteins, Cernunnos stimulates ligation of DNA ends by X4L4, and confers an ability to join mismatched ends and protruding single strands, albeit at low efficiency (5
). All these results suggest that Cernunnos is a core factor in the end joining repair complex. The finding that alignment-based gap filling in whole-cell extracts is completely dependent on Cernunnos () suggests that, in addition to stimulating ligase IV, Cernunnos is also important for DNA end alignment.
Previously, we (17
) and others (18
) have shown that in extracts of CHO (hamster) cells, end joining events requiring gap filling are completely dependent on Ku, while resection-based joining events are not. We also showed directly that X4L4 is required for gap filling, both in hamster whole-cell extracts (19
), and in human nuclear extracts (9
); furthermore, at least in the hamster extracts, XRCC4 alone could not substitute for the X4L4 complex. Given that these various extract systems show the same specificity in gap filling and the same requirement for either polλ or polμ, it appears reasonably certain that they share the same basic gap filling process. Thus, from all these data we infer that gap filling for nonhomologous end joining requires the full complex of Ku, XRCC4, DNA ligase IV and Cernunnos. The requirement for Cernunnos in gap filling is consistent with a recent proposal that end alignment is promoted by assembly of a filament of alternating homodimers of Cernunnos and XRCC4 (3
). In principle, the Cernunnos dependence of gap filling could be due to more specific interactions between Cernunnos and polλ or polμ, but gel mobility shift experiments indicate that Ku and X4L4 are sufficient to efficiently recruit either polymerase to DNA ends (20–22
). Nevertheless, a model in which Cernunnos is required for proper positioning of the polymerase within the repair complex, rather than for end alignment as such, remains a formal possibility.
The polymerase complementation results () are consistent with previous work showing that polλ and polμ have distinct but overlapping specificities. Either polymerase can efficiently fill in one-base gaps in aligned DSB ends (22
), but only polλ can fill longer gaps (9
) and only polμ can fill gaps with no base pairing, for example, when noncomplementary single-base 3′ overhangs are present on both ends (23
). Nevertheless, as shown previously with HeLa nuclear extracts (9
), immunodepletion of polλ almost completely eliminates gap filling, suggesting that the level of polμ in the extracts is too low to support robust gap filling. However, we previously found that the activity of polμ (but not polλ) was substantially reduced when the recombinant enzyme was mock-depleted from solution with pre-immune rabbit IgG (9
), and thus it is possible that polμ in the extract is more susceptible than polλ to nonspecific loss or inactivation during the extraction procedure.
Previous studies with purified DNA-PKcs, Ku, Artemis, XRCC4 and ligase IV indicate that all of these proteins except ligase IV are extensively phosphorylated by DNA-PK when they are incubated together in the presence of DNA ends (24
). However, only phosphorylation of DNA-PKcs itself has been shown to be important for end joining in cells, and phosphorylation of XRCC4 and Ku is apparently dispensable (24
). Our results with mutant Cernunnos proteins not only confirm a previous report that S245A and S251A mutations do not affect repair efficiency (14
), but also indicate that they do not affect repair fidelity.
As yet, we have been unable to detect gap filling on aligned DSB ends when the partially complementary (−ACG/−ACG) DSB substrate is incubated with the combination of Ku, DNA-PKcs, X4L4, Cernunnos, and polλ, either with or without inositol hexakisphosphate (25
), even though we have shown that each of these recombinant proteins is able to complement gap filling and end joining of the same substrate in the corresponding repair-deficient extracts (8
). Although it has been reported that alignment-based gap filling and ligation can be achieved with only purified recombinant Ku, X4L4 and polλ or polμ, this reaction requires high concentrations of substrate, as well as polyethylene glycol to promote macromolecular crowding. Moreover, these reactions are inhibited by Cernunnos as well as DNA-PKcs, both of which are required for the same reactions in cell extracts (23
). Thus, while it is difficult to rigorously eliminate the possibility that there is some artifact associated with combining multiple recombinant proteins, the simplest explanation of the available data is that there are additional components in cell extracts which, even if they are not part of the core end-joining complex, are nevertheless required for it to function properly.