Abstract
Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys(63)-linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2·phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules.
Highlights
The DNA repair process in eukaryotic cells is an indispensible life process responsible for maintaining the fidelity of the genome (1, 2)
For RAP80-(33–131), two-dimensional 1H-15N NMR spectra, and quantitative chemical shift analyses were used to derive per residue flexibility and main chain secondary structure, and indicate that the N-terminal region of RAP80 consists of three independent domains
Similar to Ub binding to the tandem Ub interacting motifs (UIM) (29), the RAP80 SUMO interacting motif (SIM) binds SUMO-2 independently
Summary
Ubiquitin; UIM, ubiquitin interacting motif; SUMO, small ubiquitin like modifier; SIM, SUMO interacting motif; CK2, casein kinase 2; DSS, 4,4-dimethyl-4-silapentane-1-sulfonic acid; PDB, Protein Data Bank; HSQC, heteronuclear single quantum coherence. The orientation of the  strand has been observed in both parallel and antiparallel conformations depending on the specific SIM sequence and SUMO isoform (19). This is believed to result from the distribution of negatively charged residues adjacent to the hydrophobic SUMO-interacting module from the SIM. This region possesses serine residues that are typically phosphorylation sites, and play a role in determining SUMO isoform preference (17, 18). We determined the first structure of SUMO-2 bound to a phosphorylated SIM, which in conjunction with measurement of the thermodynamics and kinetics of SUMO-2 binding for the phosphorylated and non-phosphorylated states of RAP80, provide insight into the molecular determinants underlying the SUMO-2 specificity of this critical DNA repair protein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
