The ubiquitin-binding zinc finger (UBZ) domain of human DNA Y-family polymerase (pol) g is important in the recruitment of the polymerase to the stalled replication machinery in translesion synthesis. Here, we report the solution structure of the pol g UBZ domain and its interaction with ubiquitin. We show that the UBZ domain adopts a classical C 2 H 2 zinc-finger structure characterized by a bba fold. Nuclear magnetic resonance titration maps the binding interfaces between UBZ and ubiquitin to the a-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single a-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol g UBZ domain represents a novel member of the C 2 H 2 zinc finger family that interacts with ubiquitin to regulate translesion synthesis.

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-␣-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K i of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.

SUMMARY Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of the Y-family polymerases (pol) ι and Rev1 at the stalled replication machinery is mediated by the ubiquitin-binding motifs (UBMs) of the polymerases and enhanced by PCNA monoubiquitination. We report the solution structures of the C-terminal UBM of human pol ι and its complex with ubiquitin. Distinct from other ubiquitin-binding domains, the UBM binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A of ubiquitin abolishes UBM binding. Human pol ι contains two functional UBMs, both of which contribute to replication foci formation. In contrast, only the second UBM of Saccharomyces cerevisiae Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations disrupting the UBM-ubiquitin interaction also impair the accumulation of pol ι in replication foci and Rev1-mediated DNA damage tolerance in vivo.