How dividing mammalian cells overcome blocks to DNA replication by DNA damage, depleted nucleotide pools, or template-bound proteins is unclear. Here, we show that the response to blocked replication requires BRCA2, a suppressor of human breast cancer. By using two-dimensional gel electrophoresis, we demonstrate that Y-shaped DNA junctions at stalled replication forks disappear during genome-wide replication arrest in BRCA2-deficient cells, accompanied by double-strand DNA breakage. But activation of the replication checkpoint kinase Chk2 is unaffected, defining an unexpected function for BRCA2 in stabilizing DNA structures at stalled forks. We propose that in BRCA2 deficiency and related chromosomal instability diseases, the breakdown of replication forks, which arrest or pause during normal cell growth, triggers spontaneous DNA breakage, leading to mutability and cancer predisposition. Received July 11, 2003; revised version accepted October 28, 2003. Germline mutations in BRCA2 cause breast, ovarian, and other cancers with a penetrance estimated at 85% by age 70 years (Nathanson et al. 2001). Although the precise activities of BRCA2 protein relevant to tumor suppression are uncertain (Scully and Livingston 2000), lossof-function mutations cause spontaneous instability of chromosome structure in dividing cells (Gretarsdottir et al. 1998;Patel et al. 1998), reminiscent of inherited diseases, such as Bloom syndrome and Fanconi anemia , also associated with cancer predisposition. Notably, the chromosomal instability induced by BRCA2 disruption frequently triggers aberrations thought to arise during replication of a damaged template (Scully and Livingston 2000; Venkitaraman 2000), including chromatid breaks and triradial and quadriradial chromosomes ). These structural abnormalities are characterized by inappropriate genetic exchanges between nonhomologous chromosomes, causing gross chromosomal rearrangements (GCRs) such as translocations or deletions (Yu et al. 2000), often found in human cancer cells. Thus, BRCA2 serves as a "caretaker" of chromosomal integrity through participation in processes linked to DNA replication. But what these BRCA2-dependent processes may be is unknown.In yeast models, spontaneous chromosomal breaks and GCRs occur in mutant strains deficient in the response to stalled or incomplete DNA replication (Chen and Kolodner 1999;Myung et al. 2001). Circumstantial evidence connects BRCA2 with this response in mammalian cells. Although it does not usually colocalize with components of the mammalian replication machinery, BRCA2 moves into PCNA-containing nuclear foci when replication is blocked (Chen et al. 1998). These findings hint at the involvement of BRCA2 in processes activated by replication inhibition. Here, we report a novel role for BRCA2. Results and Discussion Disappearance of replication forks after replication stallingHydroxyurea (HU) mimics the effects of genome-wide blocks to replication (Enoch et al. 1992;Zhao et al. 1998;Alcasabas et al. 2001) by inhibiting ribonucleotide r...

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms' tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both −KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.

The Wilms' tumour suppressor gene WT1 is essential for the normal development of the genitourinary system. It appears to play a role in both transcriptional and post-transcriptional regulation of certain cellular genes. However, the mechanisms behind WT1 function are not clearly understood despite the identification of numerous potential target genes and the isolation of several WT1-binding proteins. This study therefore sets out to identify other WT1-associating proteins to help to unravel how WT1 interacts with the cellular machinery. We report the identification of a novel human WT1-associating protein, WTAP, which was isolated using the yeast two-hybrid system. Both in vitro and in vivo assays have shown that the interaction between WTAP and WT1 is specific and occurs endogenously in cells. The mouse homologue of WTAP was isolated and found to be >90% conserved at the nucleotide and protein levels. The human and mouse genes were mapped using fluorescence in situ hybridization to regions in chromosomes 6 (which is thought to harbour a tumour suppressor gene) and 17, respectively. The expression pattern of WTAP was investigated and shown to be ubiquitous, perhaps reflecting a housekeeping role. WTAP is a nuclear protein, which like WT1 localizes throughout the nucleoplasm as well as in speckles and partially co-localizes with splicing factors. Although the significance of this interaction is not yet known, WTAP promises to be an interesting WT1-binding partner.

HPV16 is a human tumour virus encoding two principal oncoproteins, E6 and E7. Expression of E7 can induce DNA synthesis in quiescent cells and this property coincides with its ability to bind to the cell proteins pRb and p107. As these cell proteins are regulators of the transcription factor E2F, we have investigated whether the interaction with E7 could result in induction of cell cycle regulated genes. We show that B‐myb, whose induction at the G1/S boundary is regulated by release from E2F mediated transcriptional repression, is a target for transcriptional activation by E7 and is the first E7 responsive cell gene to be identified. E7 transactivation leads to both inappropriate transcription of B‐myb during G1 and constitutive over‐expression in cycling cells. B‐Myb plays an essential role in cell cycle progression, and activation by E7 is likely to contribute to the mitogenic activity of the viral oncoprotein. Regulation of the B‐myb promoter in NIH3T3 cells correlates with binding of distinct p107‐containing complexes at the E2F binding site, and analysis of E7 mutants confirms that B‐myb transcription in these cells is regulated through interactions with p107 rather than pRb. These results provide the first example of a potentially specific role for p107 in the regulation of the cell cycle.