Cellular responses to DNA damage involve the relocalization of checkpoint proteins to DNA double-strand breaks (DSBs). The fission yeast checkpoint mediator protein Crb2, a homolog of mammalian 53BP1, forms ionizing radiation-induced nuclear foci (IRIF). The IRIF formation by Crb2 requires histone H2A C-terminal phosphorylation and H4-K20 methylation. However, the relevance of Crb2 relocalization is uncertain, because neither histone modification is required for a checkpoint response. Here we show that these histone modifications cooperate in the same Crb2 recruitment pathway, which also requires the Tudor and BRCT motifs in Crb2. In the absence of these histone modifications, an alternative recruitment pathway is sufficient for checkpoint activation and accumulation of Crb2 at a persistent DSB generated by HO endonuclease. This parallel pathway requires a cyclin-dependent kinase phosphorylation site in Crb2 that mediates an association with another BRCT protein Cut5 (the TopBP1 homolog), which also accumulates at HO-induced DSBs. We propose that such dual recruitment mechanisms may be a common feature of DNA damage checkpoint mediators.[Keywords: Schizosaccharomyces pombe; chromatin; histone code; histone lysine methyltransferase; genome stability; cell cycle] Supplemental material is available at http://www.genesdev.org.

Prestin, a membrane protein that is highly and almost exclusively expressed in the outer hair cells (OHCs) of the cochlea, is a motor protein which senses membrane potential and drives rapid length changes in OHCs. Surprisingly, prestin is a member of a gene family, solute carrier (SLC) family 26, that encodes anion transporters and related proteins. Of nine known human genes in this family, three (SLC26A2, SLC26A3 and SLC26A4) are associated with different human hereditary diseases. The restricted expression of prestin in OHCs, and its proposed function as a mechanical amplifier, make it a strong candidate gene for human deafness. Here we report the cloning and characterization of four splicing isoforms for the human prestin gene (SLC26A5a, b, c and d). SLC26A5a is the predominant form of prestin whereas the others showed limited distribution associated with certain developmental stages. Based on the functional importance of prestin we screened for possible mutations involving the prestin gene in a group of deaf probands. We have identified a 5'-UTR splice acceptor mutation (IVS2-2A>G) in exon 3 of the prestin gene, which is responsible for recessive non-syndromic deafness in two unrelated families. In addition, a high frequency of heterozygosity for the same mutation was observed in these subjects, suggesting the possibility of semi-dominant influence of the mutation in causing hearing loss. Finally, the observation of this mutation only in the Caucasian probands indicated an association with a specific ethnic background. This study thereby reveals an essential function of prestin in human auditory processing.

Mutations in GJB2, encoding gap junction beta 2 protein (connexin 26), are responsible for the commonest form of non-syndromic recessive deafness in many populations. It has been reported recently that the most common 35delG mutation in GJB2 is exceptionally low in Japanese and Korean populations, but another deletion, 235delC, is relatively frequent. Since the Chinese constitute approximately one fifth of the global population, the frequency of GJB2 mutations in the population has important implications for understanding worldwide causes of genetic deafness. To determine whether GJB2 mutations are an important cause of deafness in Chinese, we conducted mutation screening for GJB2 in 118 deaf Chinese probands, including 60 from simplex and 58 from multiplex families with non-syndromic deafness, and 150 normal hearing Chinese controls. Four mutations, including 235delC, 299-300delAT, V37I, and 35delG, were found in the patients. Thirty-nine percent of the probands had a GJB2mutation. Of the 118 probands, 19 carried two definitely pathogenic mutations: three among the 58 multiplex cases (5.2%) and 16 among the 60 simplex cases (26.7%). Twenty-seven probands (22.9%) were found to carry only single GJB2 mutations. None of them had mutations in exon 1 of GJB2 and or the 342-kb deletion of GJB6. The 235delC mutation was the most prevalent mutation (20.3% of alleles), accounting for 81% of the pathologic alleles in multiplex cases and 67% in simplex cases. Analysis of the affected haplotypes in the patients with the homozygous 235delC mutation yielded evidence for a single origin of the mutation. The carrier frequency of the 235delC mutation in control subjects with normal hearing was 1.3%. The 35delG mutation was only noted as a heterozygous change in two simplex cases (1.2% of alleles). These results indicated that mutations in GJB2 are a major cause of inherited and sporadic congenital deafness in the Chinese population. The 235delC mutation, rather than 35delG, is the most common mutation found in the Chinese deaf population. Our data support the view that specific combinations of GJB2 mutation exist in different populations.

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.Eukaryotic cells respond to DNA damage by activating checkpoint signaling pathways conserved from yeast to humans (26,51). Current models of DNA damage checkpoint signaling envision distinct groups of sensor, adapter, and effector proteins acting in a sequential manner to effect checkpoint responses (20,35). Sensor proteins act at the top of the pathways by recognizing the DNA lesions. Adapter proteins then transduce the signal from the sensors to downstream effectors (Table 1).Three checkpoint protein complexes have been suggested to play roles in sensing DNA damage. The components of these complexes in fission yeast are the "checkpoint Rad proteins," including Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1. The Rad3-Rad26 complex is a protein kinase orthologous to budding yeast Mec1-Lcd1 and mammalian ATR-ATRIP (8). Rad17 associates with four replication factor C (RFC) subunits to form a pentameric complex that is thought to load the ring-shaped Rad1-Rad9-Hus1 "sliding clamp" onto DNA (11,39,46). Recent studies showed that the ATR-ATRIP complex and the checkpoint sliding clamp are recruited to the chromatin at the sites of DNA damage through distinct mechanisms (16,20,34,36,52). The independent recruitment of these two complexes at sites of DNA damage suggests that proper recognition of DNA damage may require multiple checkpoint sensors with distinct functions.In fission yeast, the checkpoint Rad proteins are required for both the replication checkpoint that senses stalled replication forks and activates the effector kinase Cds1 (Chk2) and the DNA damage checkpoint that activates the effector kinase Chk1 (6, 32). The different outputs of the two checkpoint pathways are likely to be determined by adapter checkpoint proteins, which provide specificity to the checkpoint responses initiated by a common set of sensor proteins (41). The fission yeast adapter protein involved in the DNA damage checkpoint is Crb2, which shares sequence similarity in its C-terminal tandem BRCT repeats with budding yeast Rad9 and mammalian 53BP1 and BRCA1 (37, 49). ...

We report a large Chinese family with X-linked postlingual nonsyndromic hearing impairment in which the critical linkage interval spans a genetic distance of 5.41 cM and a physical distance of 15.1 Mb that overlaps the DFN2 locus. Mutation screening of the PRPS1 gene in this family and in the three previously reported DFN2 families identified four different missense mutations in PRPS1. These mutations result in a loss of phosphoribosyl pyrophosphate (PRPP) synthetase 1 activity, as was shown in silico by structural analysis and was shown in vitro by enzymatic activity assays in erythrocytes and fibroblasts from patients. By in situ hybridization, we demonstrate expression of Prps1 in murine vestibular and cochlea hair cells, with continuous expression in hair cells and postnatal expression in the spiral ganglion. Being the second identified gene associated with X-linked nonsyndromic deafness, PRPS1 will be a good candidate gene for genetic testing for X-linked nonsyndromic hearing loss.