Human SNM1 protein is a single-strand 5'-3' exonuclease acting in DNA crosslink repair. J. Hejna, S. Philip, C. Faulkner, A. Hemphill, J. Ott, R. Moses. Molec & Med Genetics, L103, Oregon Health Sciences Univ, Portland, OR.
   DNA interstrand crosslinking agents are particularly potent DNA damaging agents because they constrain DNA topology, impeding replication and transcription. Interstrand crosslinks (ICLs) must be removed by a multi-step process, and in yeast three independent pathways are required for optimal repair of ICLs. The yeast SNM1 protein is a 5'-3' exonuclease, operates in the Rad3 pathway, and is needed for normal ICL repair. In human cells, five SNM1 homologs (SNM1, SNM1B, SNM1C/Artemis, ELAC2, and CPSF73) have been identified, raising the question of whether the enzymological function of the protein has been conserved, or whether different homologs have acquired specialized functions. In order to characterize hSNM1 activity, recombinant protein was expressed and purifed from KC167 insect cells. It possesses a strong 5'-3' exonuclease activity, using a single-stranded DNA oligonucleotide substrate. A 5'-phosphate was strictly required for the hSNM1 exonuclease. Gel filtration indicated that hSNM1, like ySNM1, functions as a monomer. Mutation of a conserved aspartate residue (D736 and D252, in hSNM1 and ySNM1, respectively) within the -CASP domain abrogated the activity in both proteins. Whereas ySNM1 efficiently digests both single-stranded and double-stranded substrates, hSNM1 shows a marked preference for single-stranded substrates. Depletion of hSNM1 by siRNA leads to increased chromosomal breaks and radial formations, and decreased cell survival after treatment with mitomycin C. We conclude that hSNM1 is a 5'-3' exonuclease required for normal ICL repair.