Human DNA ligase IV is able to use NAD(+) as an alternative adenylation donor for DNA ends ligation

All the eukaryotic DNA ligases are known to use adenosine triphosphate (ATP) for DNA ligation. Here, we report that human DNA ligase IV, a key enzyme in DNA double-strand break (DSB) repair, is able to use NAD(+) as a substrate for double-stranded DNA ligation. In the in vitro ligation assays, we show that the recombinant Ligase IV can use both ATP and NAD(+) for DNA ligation. For NAD(+)-mediated ligation, the BRCA1 C-terminal (BRCT) domain of Ligase IV recognizes NAD(+) and facilitates the adenylation of Ligase IV, the first step of ligation. Although XRCC4, the functional partner of Ligase IV, is not required for the NAD(+)-mediated adenylation, it regulates the transfer of AMP moiety from Ligase IV to the DNA end. Moreover, cancer-associated mutation in the BRCT domain of Ligase IV disrupts the interaction with NAD(+), thus abolishes the NAD(+)-mediated adenylation of Ligase IV and DSB ligation. Disrupting the NAD(+) recognition site in the BRCT domain impairs non-homologous end joining (NHEJ) in cell. Taken together, our study reveals that in addition to ATP, Ligase IV may use NAD(+) as an alternative adenylation donor for NHEJ repair and maintaining genomic stability.