Several laboratories have made dedicated attempts to identif

Several laboratories have made dedicated attempts to identify A-NHEJ factors. In particular, a brute-force nuclear extract fractionation protocol identified LIGIII (DNA ligase III; [12]), heretofore known only for its role in BER (base excision repair), as the candidate ligase required for A-NHEJ [16]. Using guilt-by-association as a scientific rationale, PARP1 (poly (ADP-ribose) polymerase 1) and XRCC1 (X-ray cross complementing gene 1), two proteins known to interact with LIGIII during BER, were subsequently identified as also being involved in A-NHEJ [13], [17], [18]. PARP1 is presumed to compete with Ku for binding to broken DNA ends thereby dictating pathway choice [13], [18] whereas XRCC1 appears to act as a chaperone for LIGIII [19]. Additional factors have also been implicated in A-NHEJ. Thus, CtIP (C-terminal interacting protein) and the MRN (Mre11:Rad50:Nbs1) complex – factors known to be involved in the end resection events required for HR – have also been implicated in the end resection steps of A-NHEJ [20], [21], [22], [23], [24]. If the factors needed for A-NHEJ are not completely defined and the A-NHEJ reaction mechanism nebulous, it is also fair to say that the biological role(s) of A-NHEJ is even more poorly understood. Most of the current interest in A-NHEJ, however, stems from its implicated use in the chromosomal translocations that are present in cancer cells. Sequencing of human cancer genomes has revealed that many [25], [26], [27], albeit not all [28] chromosomal translocations have microhomology at their breakpoint junctions, which implicates A-NHEJ in their genesis. This hypothesis gained support from work in which LIGIII conditionally-null murine Coumarin showed decreased translocation frequencies and reduced microhomology usage [29], [30], [31], [32]. An additional biological process where A-NHEJ has been implicated is in the random insertion events associated with rAAV (recombinant adeno-associated virus)-mediated gene targeting. Although rAAV can mediate high frequency gene targeting, the majority of the viral integration events still occur randomly [33]. Moreover, our laboratory has reported that a reduction in the C-NHEJ genes Ku70 [34] and LIGIV [35] had almost no impact on the random rAAV integration rate – implying that these events may be mediated instead by A-NHEJ. In summary, although A-NHEJ was a neglected subject for many years, in the past decade it has proven itself to be an increasingly interesting and biologically relevant topic. A key feature of A-NHEJ is its dependence on LIGIII [16]. Unlike the other ligases, LIGIII is molecularly heterogeneous [12], [36], [37]. Thus, alternative translation initiation generates mitochondrial and nuclear forms of LIGIII, which either contain or lack a MLS (mitochondrial localizing sequence), respectively [36]. The existence of LIGIII isoforms implies diverse functional roles for LIGIII. One experimental approach to unraveling the complexity of LIGIII is to generate a LIGIII-deficient model system, which has already been accomplished in the chicken cell line, DT40 [38], and in the mouse [30], [32], [39]. In these systems the gene is essential due to its presumed requirement for mitochondrial DNA replication. Moreover, in LIGIII conditionally-null mice no obvious nuclear DNA repair phenotypes could be detected [30], [32]. The extrapolation of these studies to humans is unfulfilled as neither LIGIII patients nor LIGIII-deficient human cell systems have been described.