Structural Insights into Recognition of MDC1 by TopBP1 in DNA Replication Checkpoint Control

SummaryActivation of the DNA replication checkpoint by the ATR kinase requires protein interactions mediated by the ATR-activating protein, TopBP1. Accumulation of TopBP1 at stalled replication forks requires the interaction of TopBP1 BRCT5 with the phosphorylated SDT repeats of the adaptor protein MDC1. Here, we present the X-ray crystal structures of the tandem BRCT4/5 domains of TopBP1 free and in complex with a MDC1 consensus pSDpT phosphopeptide. TopBP1 BRCT4/5 adopts a variant BRCT-BRCT packing interface and recognizes its target peptide in a manner distinct from that observed in previous tandem BRCT- peptide structures. The phosphate-binding pocket and positively charged residues in a variant loop in BRCT5 present an extended binding surface for the negatively charged MDC1 phosphopeptide. Mutations in this surface reduce binding affinity and recruitment of TopBP1 to γH2AX foci in cells. These studies reveal a different mode of phosphopeptide binding by BRCT domains in the DNA damage response

MDC1 collaborates with TopBP1 in DNA replication checkpoint control

The DNA damage checkpoint protein MDC1 also interacts with TopBP1 to promote DNA replication checkpoint control

Controlled synthesis of highly-branched plasmonic gold nanoparticles through peptoid engineering

In nature, specific biomolecules interacting with mineral precursors are responsible for the precise production of nanostructured inorganic materials that exhibit complex morphologies and superior performance. Despite advances in developing biomimetic approaches, the design rules for creating sequence-defined molecules that lead to the synthesis of inorganic nanomaterials with predictable complex morphologies are unknown. Herein we report the design of sequence-defined peptoids for controlled synthesis of highly branched plasmonic gold particles. By engineering peptoid sequences and investigating the resulting particle formation mechanisms, we develop a rule of thumb for designing peptoids that predictively enabled the morphological evolution from spherical to coral-shaped nanoparticles. Through a combination of hyperspectral UV-Vis extinction microscopy and three-photon photoemission electron microscopy, we demonstrate that the individual coral-shaped gold nanoparticles exhibit a plasmonic enhancement as high as 105-fold. This research significantly advances our ultimate vision of predictive bio-inspired materials synthesis using sequence-defined synthetic molecules that mimic proteins and peptides

SUMO Modification Enhances p66-Mediated Transcriptional Repression of the Mi-2/NuRD Complex

Human p66α and p66β are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66α and one in p66β. Both p66α and p66β are SUMO-modified in vivo: p66α at two sites (Lys-30 and Lys-487) and p66β at one site (Lys-33). Expression of SUMO1 enhanced the transcriptional repression activity of Gal-p66α and Gal-p66β. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase resulted in a significant decrease of the transcriptional repression of p66α and p66β. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 were found to bind to both p66α and p66β in vivo. Most of the interactions were not affected by the SUMO site mutations in p66α or p66β, with two exceptions. HDAC1 binding to p66α was lost in the case of a p66αK30R mutant, and RbAp46 binding was reduced in the case of a p66βK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation

Nuclear distribution of p66α and p66β depends on CR2 and K149 for p66α

<p><b>Copyright information:</b></p><p>Taken from "p66α and p66β of the Mi-2/NuRD complex mediate MBD2 and histone interaction"</p><p>Nucleic Acids Research 2006;34(2):397-406.</p><p>Published online 13 Jan 2006</p><p>PMCID:PMC1331983.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> NIH3T3 cells were transfected with the indicated constructs coding for various EGFP-fused p66-proteins. Cells were stained with ‘Hoechst DNA stain’ and phase contrast images (left), Hoechst stain fluorescence (middle) and EGFP fluorescence (right) was visualized

PTIP associates with Artemis to dictate DNA repair pathway choice.

PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1(-/-)53BP1(-/-) cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway

Nuclear distribution of p66α and p66β depends on the presence of MBD2

<p><b>Copyright information:</b></p><p>Taken from "p66α and p66β of the Mi-2/NuRD complex mediate MBD2 and histone interaction"</p><p>Nucleic Acids Research 2006;34(2):397-406.</p><p>Published online 13 Jan 2006</p><p>PMCID:PMC1331983.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> MBD2+/+ wild-type or MBD2−/− knock-out fibroblast cells were transfected with constructs coding for the indicated EGFP-fused p66-proteins. Images were taken as in