Biochemical and cell biological experiments implicate the nuclear Lig3-XRCC1 complex in single-strand break repair, short patch base excision repair, and nucleotide excision repair1
. Lig3 and XRCC1 interact via C-terminal BRCT domains found in each protein7
. This interaction is important for the stability of Lig37
and the recruitment of Lig3 to DNA damage foci8
. Purified Lig3-XRCC1 is proficient at nick sealing in vitro9
, and the complex associates with several other proteins involved in single-strand break repair, including Parp110
, aprataxin, and TDP11
Lig3 also has a mitochondrial form due to an alternative translation start site, which results in a mitochondrial leader sequence (MLS)11
. Mammals differ in this respect from budding yeast, where the Lig1 homolog, Cdc9, is the mitochondrial DNA ligase12
. In mitochondria, Lig3 appears to act independently of XRCC1, as XRCC1 is not present in this organelle13
. Disruption of the Lig3
gene, like Xrcc1
, results in early embryonic lethality in the mouse2,5
, and Lig3
null cell lines could not be established from these animals2
. The similar timing of lethality of Lig3
null embryos suggests that death could result from similar phenotypic consequences related to Lig3 nuclear functions in DNA repair. Alternatively, or in addition, the mitochondrial function of Lig3 may be critical for survival.
To determine whether Lig3
is an essential gene due to its nuclear and/or mitochondrial function, we developed a pre-emptive complementation strategy in mouse ES cells (). A Lig3KO/cKOneo+
cell line was constructed which contains one conditional Lig3
allele with an intronic Neo
selection marker and LoxP
sites flanking exons 6 and 14 and a second allele in which these exons were already removed by Cre recombinase (; Supplementary Fig. 1
). These exons encode part of the DNA binding domain and the catalytic core of the protein. Cre recombinase was expressed in the Lig3KO/cKOneo+
cells, and 145 clones were replica-plated in media with or without G418. No G418 sensitive clones (i.e., Lig3KO/KO
) were obtained (), consistent with the requirement for Lig3 for cellular viability. We then stably integrated transgenes expressing wild-type, mitochondrial, or nuclear Lig3; the nuclear (NucLig3) version lacked the MLS, and the mitochondrial (MtLig3) version contained the MLS but was mutated at the nuclear translation initiation site (M88T) (; Supplementary Fig. 2
). GFP fusions of these proteins were also tested (Supplementary Fig. 3a
Mitochondrial Lig3 activity is critical for cellular viability
To determine which Lig3
transgenes permit the survival of cells deleted for endogenous Lig3
, Cre recombinase was used to transform Lig3KO/cKOneo+
cells to Lig3KO/KO
cells. A large fraction of the post-Cre clones expressing wild-type Lig3 or MtLig3 were G418 sensitive (34 to 50%), whereas no G418 sensitive clones were obtained with NucLig3 (; Supplementary Fig. 4
). We confirmed that G418 sensitive cells were Lig3KO/KO
() and that endogenous Lig3 was no longer present, with the only Lig3 present in the cells expressed from the transgene (). Thus, cellular viability requires mitochondrial Lig3. To determine whether DNA ligase activity was essential for cell survival, we introduced a K508V mutation that abolishes ligase adenylylation and nick-sealing1
into MtLig3. No G418 sensitive clones were derived from 4 independent transgenic cell lines (), demonstrating that the requirement for mitochondrial Lig3 depends on its ligase activity.
BRCT domains are frequently involved in protein-protein interactions, and the BRCT domain of Lig3 is known to interact with XRCC17
. However, as XRCC1 is not found in mitochondria13
, the role of the BRCT domain for mitochondrial function of Lig3 is uncertain. Loss of the BRCT domain had no effect on the presence of Lig3 in mitochondria (Lig3-ΔBRCT and MtLig3-ΔBRCT; Supplementary Fig. 3a
; data not shown). Lig3KO/KO
clones expressing Lig3-ΔBRCT or MtLig3-ΔBRCT () were recovered as a substantial fraction of clones (39 to 49%; ), indicating that the BRCT domain is not required for viability. Thus, MtLig3 does not have a partner protein bound to its BRCT domain that is essential for its function.
Mitochondrial DNA ligase activity can be provided by a variety of DNA ligases
A unique feature of Lig3 compared with the other mammalian DNA ligases is a ZnF at its N-terminus. The Lig3 ZnF interacts with Parp110
, and this interaction is reported to be important for the association of Lig3 with mitochondrial DNA (mtDNA)14
. Biochemical studies have shown that the ZnF promotes DNA nick recognition15
and intermolecular ligation16
. Nonetheless, Lig3KO/KO
clones expressing Lig3-ΔZnF or MtLig3-ΔZnF () were efficiently recovered after Cre expression (37 to 38%; ).
Our results reveal that the catalytic activity of Lig3 is critical for cell survival, but that the ZnF and BRCT domains, which interact with various proteins, are dispensable, raising the question whether Lig3 itself is critical for mitochondrial function, or whether another DNA ligase would substitute. As the Lig1 homolog in yeast provides mitochondrial ligase function12
, we provided an MLS to murine Lig1 (). GFP-tagged MtLig1, but not wild-type Lig1, was localized to mitochondria (Supplementary Fig. 3c
), as expected. Stable Lig3KO/cKOneo+
cell lines expressing MtLig1, but not wild-type Lig1, could be efficiently converted to Lig3KO/KO
(35%; ). MtLig1 Lig3KO/KO
clones were devoid of Lig3, and expressed instead a larger Lig1 protein due to the GFP tag (). Thus, targeting Lig1 to mitochondria circumvents the viability requirement for Lig3, allowing the creation of Lig3
null cells. In this way, the DNA ligase repertoire of mammalian cells is converted to that of yeast.
Loss of Lig3 is not associated with sensitivity to several DNA damaging agents or with increased sister-chromatid exchange
Given that ZnF and BRCT-truncated forms of Lig3 and MtLig1 could rescue Lig3KO/KO
cells, we investigated their proficiency in mtDNA maintenance and repair. The mtDNA copy number was maintained as well (or better) in these cell lines as in wild-type Lig3-expressing cells (), indicating that cells expressing these altered ligases are competent to replicate their mtDNA during continued passage. A long-range quantitative PCR assay17
was performed to assess the mitochondrial base excision repair capacity of these cells in response to oxidative damage, and these altered ligases were similarly proficient in repairing mtDNA lesions compared to wild-type Lig3-expressing cells ().
At 298 amino acids, Chlorella
virus DNA ligase (ChVLig) is the smallest eukaryal ligase known, consisting solely of a catalytic core3
. We expressed ChVLig and a modified form containing an MLS, MtChVLig (), and found that expression of either allowed deletion of Lig3
from the mouse genome (). It is conceivable that ChVLig contains an internal sequence that allows translocation into mitochondria18
Thus, a minimal ATP-dependent ligase, devoid of auxiliary domains, rescues the survival of Lig3 null mammalian cells. Further, Lig3 null cells rescued by MtChVLig were proficient at mtDNA maintenance () and repair ().
Whereas ATP-dependent ligases are widespread, ligases that use NAD+ as a cofactor are usually restricted to bacteria1
. E. coli
DNA ligase, LigA, is NAD+-dependent and has a distinctive domain organization compared with mammalian ligases1
. LigA and a modified form with an MLS () were expressed from transgenes, and like ChVLig, both forms were found to allow the survival of Lig3 null cells (). Hence, there is no essential functional distinction between NAD+ and ATP-dependent ligases in the mammalian mitochondria, akin to swaps of NAD+ and ATP-dependent ligases performed in bacteria19
We demonstrated that nuclear Lig3 is not required for cell survival, as MtLig1 Lig3KO/KO
cells are null for Lig3
. To impair nuclear localization of MtLig1, we removed the Lig1 nuclear localization signal, creating MtLig1-ΔNLS Lig3KO/KO
cells (; Supplementary Fig. 3c
). MtLig1-ΔNLS, like MtLig1, was expressed at a substantially lower level than endogenous Lig1 (). As a complementary approach, we also created Lig3KO/KO
cells expressing MtLig3-ΔBRCT-NES, whose interaction with XRCC1 is abrogated and which is excluded from the nucleus by addition of a potent nuclear export signal (NES)21
(; ; Supplementary Fig. 3b
To assess the nuclear role of Lig3, we tested the sensitivity of Lig3
; MtLig1-ΔNLS) and nuclear Lig3-deficient (Lig3KO/KO
; MtLig3-ΔBRCT-NES) cells to a variety of DNA damaging agents. XRCC1-deficient cells are highly sensitive to alkylating agents like methyl methanesulfonate (MMS)4,5,6
. If the interaction of XRCC1 with Lig3 is relevant to base excision repair, cells without nuclear Lig3 would also be expected to be sensitive to MMS; however, we found that these cells were no more sensitive than transgenic cells expressing wild-type Lig3 () or the parental cells (Supplementary Fig. 5
). XRCC1-deficient cells are also sensitive to agents which produce DNA single and double-strand breaks, including hydrogen peroxide and ionizing radiation4,5,6
, and to ultraviolet radiation22
. By contrast, we found that cells without nuclear Lig3 were not any more sensitive to these agents than control cells (, Supplementary Fig. 5
). Thus, Lig3 appears to be dispensable for nuclear DNA damage repair that requires XRCC1. Finally, we tested sensitivity to Parp inhibiton, which causes the accumulation of single-strand breaks23
, and found that nuclear Lig3 was also not required for resistance of cells to Parp inhibiton ().
As the ZnF domain of Lig3 has been reported to be critical for its intermolecular ligation activity16
, we also investigated whether deletion of this domain in the context of an otherwise wild-type Lig3 would impair resistance of cells to ionizing radiation. As with the other mutants, Lig3-ΔZnF Lig3KO/KO
cells were no more sensitive than control cells ().
XRCC1-deficient cells are notable for their high rate of spontaneous sister-chromatid exchange (SCE): both mouse and hamster XRCC1 mutants have ~10-fold higher SCE levels than control cells4,5
. We examined spontaneous SCEs in MtLig1 Lig3KO/KO
cells and found levels similar to control cells (). Thus, the high level of SCEs found with XRCC1 deficiency is not recapitulated with Lig3 deficiency.
The lack of Lig3 in many model organisms has limited their use to study its function. In mouse, disruption of any of the DNA ligase genes leads to embryonic death, but the most severe phenotype occurs with Lig3 disruption2,24,25
. Lig1 has been considered to be the replicative ligase1,26
, but the earlier death associated with Lig3 disruption, together with the inability to obtain viable Lig3 null cells, left open the possibility that Lig3 could have a critical role in nuclear DNA metabolism. The generation of viable and healthy Lig3
null cells by providing a mitochondrial ligase conclusively rules out an essential role for Lig3 in the nucleus.
The well-documented interaction between Lig3 and XRCC1 had suggested that Lig3 would be critical for the same nuclear DNA repair pathways as XRCC1, similar to the Lig4-XRCC4 complex in DSB repair1
. However, the lack of sensitivity of Lig3 null cells to the spectrum of DNA damaging agents that sensitize XRCC1-deficient cells, as shown here in proliferating cells and in the accompanying report in quiescent cells27
, together with a normal SCE frequency, provides strong evidence that Lig3 is not required for XRCC1-dependent nuclear DNA repair, pointing instead to a role for Lig1.
Our results demonstrate instead that Lig3 is an essential gene because of its requirement in mitochondria. However, Lig3 can be replaced in mitochondria with Lig1, the mitochondrial ligase in lower eukaryotes, with an algal viral ligase consisting solely of a catalytic core, and even the NAD+-dependent E. coli LigA. Thus, these results attest to the requirement for a functional DNA ligase, which trumps even co-factor specificity. Why vertebrates developed a requirement for Lig3 is uncertain, but given our results, the additional domains found in Lig3 do not appear to be essential for mitochondrial function, including mtDNA maintenance or repair of oxidative damage. These results underscore a surprising plasticity that mammalian cells have in their mitochondrial DNA ligase requirement.