Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress.

The ATR-mediated checkpoint is not only critical for responding to genotoxic stress but also essential for cell proliferation. The RFC-related checkpoint protein Rad17, a phosphorylation substrate of ATR, is critical for ATR-mediated checkpoint signaling and cell survival. Here, we show that phosphorylation of Rad17 by ATR is important for genomic stability and restraint of S phase but is not essential for cell survival.

Dissecting cancer pathways and vulnerabilities with RNAi.

The latest generation of molecular-targeted cancer therapeutics has bolstered the notion that a better understanding of the networks governing cancer pathogenesis can be translated into substantial clinical benefits. However, functional annotation exists for only a small proportion of genes in the human genome, raising the likelihood that many cancer-relevant genes and potential drug targets await identification. Unbiased genetic screens in invertebrate organisms have provided substantial insights into signaling networks underlying many cellular and organismal processes.

ATM-Chk2-p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency.

BRCA1 is a checkpoint and DNA damage repair gene that secures genome integrity. We have previously shown that mice lacking full-length Brca1 (Brca1(delta11/delta11)) die during embryonic development. Haploid loss of p53 completely rescues embryonic lethality, and adult Brca1(delta11/delta11)p53+/- mice display cancer susceptibility and premature aging.

Control of ribonucleotide reductase localization through an anchoring mechanism involving Wtm1.

The control of deoxyribonucleotide levels is essential for DNA synthesis and repair. This control is exerted through regulation of ribonucleotide reductase (RNR). One mode of RNR regulation is differential localization of its subunits.

The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression.

The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A.

53BP1 oligomerization is independent of its methylation by PRMT1.

p53 binding protein 1 (53BP1) participates in the repair of DNA double stranded breaks (DSBs) where it is recruited to or near sites of DNA damage. Although little is known about the biochemical functions of 53BP1, the protein possesses several motifs that are likely important for its role as a DNA damage response element. This includes two BRCA1 C-terminal repeats, tandem Tudor domains, and a variety of phosphorylation sites.

BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1-Chk1 pathway, implicating checkpoint dysfunction in microcephaly.

BRIT1 [BRCT-repeat inhibitor of hTERT expression], a repressor of human telomerase function, is implicated in cellular immortalization. Here, we find that BRIT1 acts as a regulator of both the intra-S and G2/M checkpoints. When BRIT1 expression is depleted, cells lose the ionizing radiation (IR)-induced cell cycle arrest and become IR sensitive.

Second-generation shRNA libraries covering the mouse and human genomes.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry.

A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells.

The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA (shRNA) from polymerase III promoters can be encoded in transgenes and used to produce small interfering RNAs that down-regulate specific genes. In this study, we show that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context.

A genetic screen for candidate tumor suppressors identifies REST.

Tumorigenesis is a multistep process characterized by a myriad of genetic and epigenetic alterations. Identifying the causal perturbations that confer malignant transformation is a central goal in cancer biology. Here we report an RNAi-based genetic screen for genes that suppress transformation of human mammary epithelial cells.

MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules.

We describe a highly engineered in vivo cloning method, mating-assisted genetically integrated cloning (MAGIC), that facilitates the rapid construction of recombinant DNA molecules. MAGIC uses bacterial mating, in vivo site-specific endonuclease cleavage and homologous recombination to catalyze the transfer of a DNA fragment between a donor vector in one bacterial strain and a recipient plasmid in a separate bacterial strain. Recombination events are genetically selected and result in placement of the gene of interest under the control of new regulatory elements with high efficiency.

Genetic analysis of the kinetochore DASH complex reveals an antagonistic relationship with the ras/protein kinase A pathway and a novel subunit required for Ask1 association.

DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants.

Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase.

Turnover of cyclin E is controlled by SCF(Fbw7). Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7alpha and -gamma are nuclear and the beta-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay.

Chk1 is haploinsufficient for multiple functions critical to tumor suppression.

The haploinsufficient tumor suppressor Chk1 is essential for embryonic cells, but the consequences of Chk1 loss in adult tissues are unknown. Using conditional Chk1 mice, we find that proliferating mammary cells lacking Chk1 undergo apoptosis leading to developmental defects. Conditional Chk1 heterozygosity increased the number of S phase cells and caused spontaneous DNA damage.

Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases.

The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. We describe four lines of evidence that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex.

Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation.

Claspin is a homolog of Mrc1, a checkpoint protein required for the DNA replication checkpoint in yeast. In Xenopus, phosphorylated Claspin binds to xChk1 and regulates xChk1 activation in response to replication stress. In this study, we have shown that the human homolog of Claspin is required for resistance to multiple forms of genotoxic stress including UV, IR, and hydroxyurea.

A resource for large-scale RNA-interference-based screens in mammals.

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating.

Cyclin F disruption compromises placental development and affects normal cell cycle execution.

Human cyclin F was originally isolated as a cDNA capable of suppressing the temperature sensitivity of a Saccharomyces cerevisiae cdc4-1 mutant. Its tightly regulated expression and high conservation in the evolutionary progression from amphibians to mammals suggest that it coordinates the timing of a critical cell cycle event. The fact that it contains an F box and can form an SCF (Skp1-Cul1/Cdc53-F-box) complex in vivo further suggests that it may also function in proteolysis.

Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein.

The mammalian F-box protein Fbw7 and its Caenorhabditis elegans counterpart Sel-10 have been implicated in the ubiquitin-mediated turnover of cyclin E as well as the Notch/Lin-12 family of transcriptional activators. Both unregulated Notch and cyclin E promote tumorigenesis, and inactivating mutations in human Fbw7 suggest that it may be a tumor suppressor. To generate an in vivo system to assess the consequences of such unregulated signaling, we generated mice deficient for Fbw7.

SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase.

Eukaryotic cells respond to DNA damage and stalled replication forks by activating protein kinase-mediated signaling pathways that promote cell cycle arrest and DNA repair. A central target of the cell cycle arrest program is the Cdc25A protein phosphatase. Cdc25A is required for S-phase entry and dephosphorylates tyrosine-15 phosphorylated Cdk1 (Cdc2) and Cdk2, positive regulators of cell division.

Replication protein A-mediated recruitment and activation of Rad17 complexes.

The human Rad17-Rfc2-5 and Rad9-Rad1-Hus1 complexes play crucial roles in the activation of the ATR-mediated DNA damage and DNA replication stress response pathways. In response to DNA damage, Rad9 is recruited to chromatin in a Rad17-dependent manner in human cells. However, the DNA structures recognized by the Rad17-Rfc2-5 complex during the damage response have not been defined.