Isaac A. Manke

RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly.

DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

Structure and mechanism of BRCA1 BRCT domain recognition of phosphorylated BACH1 with implications for cancer.

Germline mutations in the BRCA1 tumor suppressor gene often result in a significant increase in susceptibility to breast and ovarian cancers. Although the molecular basis of their effects remains largely obscure, many mutations are known to target the highly conserved C-terminal BRCT repeats that function as a phosphoserine/phosphothreonine-binding module. We report the X-ray crystal structure at a resolution of 1.85 Å of the BRCA1 tandem BRCT domains in complex with a phosphorylated peptide representing the minimal interacting region of the DEAH-box helicase BACH1. The structure reveals the determinants of this novel class of BRCA1 binding events. We show that a subset of disease-linked mutations act through specific disruption of phospho-dependent BRCA1 interactions rather than through gross structural perturbation of the tandem BRCT domains.

Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores

BACKGROUND In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression. RESULTS We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest. CONCLUSIONS Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.

Chk’n Out in Mitosis

No abstract available.