The mammalian family X DNA polymerases (DNA polymerases beta, lambda, mu, and TdT) contribute to base excision repair and double-strand break repair by virtue of their ability to fill short gaps in DNA. Structural information now exists for all four of these enzymes, making this the first mammalian polymerase family whose structural portrait is complete. Here we consider how distinctive structural features of these enzymes contribute to their biological functions in vivo.

DNA polymerase mu (Pol mu) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol mu's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol mu bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol mu that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol mu from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3' ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.

The receptor for activated C-kinase 1 (RACK1) is a highly conserved WD40 repeat scaffold protein found in a wide range of eukaryotic species from Chlamydymonas to plants and humans. In tissues of higher mammals, RACK1 is ubiquitously expressed and has been implicated in diverse signaling pathways involving neuropathology, cellular stress, protein translation, and developmental processes. RACK1 has established itself as a scaffold protein through physical interaction with a myriad of signaling proteins ranging from kinases, phosphatases, ion channels, membrane receptors, G proteins, IP3 receptor, and with widely conserved structural proteins associated with the ribosome. In the plant Arabidopsis thaliana, RACK1A is implicated in diverse developmental and environmental stress pathways. Despite the functional conservation of RACK1-mediated protein-protein interactionregulated signaling modes, the structural basis of such interactions is largely unknown. Here we present the first crystal structure of a RACK1 protein, RACK1 isoform A from Arabidobsis thaliana, at 2.4 Å resolution, as a C-terminal fusion of the maltose binding protein. The structure implicates highly conserved surface residues that could play critical roles in protein-protein interactions and reveals the surface location of proposed post-transcriptionally modified residues. The availability of this structure provides a structural basis for dissecting RACK1-mediated cellular signaling mechanisms in both plants and animals.Keywords: RACK1; protein signaling; scaffolding protein; WD40; propeller; protein-protein interaction; drought Scaffold proteins are uniquely poised to integrate signals from multiple pathways. Such proteins generate considerable functional diversity by mediating concomitant and/ or promiscuous interactions with a vast array of protein partners. The receptor for activated C-kinase 1 (RACK1) is a member of the WD40 repeat family of b-propeller proteins. It was discovered through its ability to function as a scaffold protein, stabilizing signaling complexes involving protein kinase C (Ron and Mochly-Rosen 1994; . In metazoans, RACK1 is now postulated to coordinate many different signal transduction pathways, ranging from cell division to ion channel regulation, by interacting with more than 80 different proteins (McCahill et al. 2002;Alfarano et al. 2005; 4 Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi