A New Pathway for DNA Cross-Link Repair
Interstrand cross-links (ICLs) are a highly toxic form of DNA damage in which the two complementary DNA strands are covalently bound together. This form of damage is difficult to repair, so animal cells have evolved the Fanconi anemia (FA) pathway specifically for this purpose. The FA pathway comprises >20 proteins, but not all of the players have been identified. In particular, the identity of the nuclease(s) that cleave on the 5′ side of the ICL is unclear. Thus, when Vanderbilt Basic Sciences investigator Ian Macara and his lab discovered a new enzyme with 5′ exonuclease activity, they quickly evaluated its potential role in ICL repair. The discovery began with the realization that a protein originally identified as a transcriptional co-activator of PPAR-γ contained a nuclease domain. Extensive studies of the activity of the protein with various potential substrates revealed it to be an exonuclease that favored substrates with a free 5′ end at the terminus of a substantial single-stranded segment. Knockout of the gene for this protein in HeLa cells using CRISPR/cas technology resulted in an accumulation of DNA damage upon exposure to ICL-inducing agents along with increased sensitivity of the cells to the toxicity of these agents. These findings led the investigators to suspect that the protein was involved in the FA pathway. However, RNAi-mediated knockdown of FA pathway proteins acted synergistically with knockout of the nuclease to increase sensitivity to ICL-inducing agents, suggesting that protein works independently of the FA pathway. A genome-wide yeast two-hybrid screen revealed that the nuclease interacts with the protein senataxin, a helicase known to be involved with DNA transcription and repair. As a result of this finding, the investigators named the nuclease SAN1 (senataxin-associated nuclease 1). The researchers identified the region of SAN1 required for its interaction with senataxin and demonstrated that deletion of this region eliminated the protein’s ability to protect cells against ICL-mediated toxicity. They also showed that knockdown of senataxin had a similar effect on sensitivity to ICL-inducing agents as knockout of SAN1. Senataxin is known to be involved in resolution of R-loops, DNA/RNA hybrid structures that accumulate when DNA damage stalls transcription. The researchers found that cells lacking SAN1 exhibited an accumulation of R-loops similar to that seen upon knockdown of senataxin. Together the results show that SAN1 is a 5′ exonuclease that works in concert with senataxin to repair ICLs through a mechanism distinct from the FA pathway and likely linked to transcription. Further work will be required to fully elucidate the other proteins involved in this pathway.The work is published in the journal Nature Communications[A. M. Andrews, et al. Nat. Comm., (2018) 9, 2592].