Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase h (polh), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase i (poli), like polh, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that polh and poli foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poli in replication foci is largely dependent on the presence of polh. Using several different approaches, we demonstrate that polh and poli interact with each other physically and that the C-terminal 224 amino acids of poli are suf®cient for both the interaction with polh and accumulation in replication foci. Our results provide strong evidence that polh targets poli to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis. Keywords: DNA polymerase/replication foci/UV light/ xeroderma pigmentosum variants Introduction DNA damage occurs ubiquitously in all cells. In order to maintain the stability of the genome, cells have evolved mechanisms not only to repair all types of DNA damage, but also to replicate DNA from which the damage has not been removed (post-replication repair). In the case of human cells, a major mechanism for carrying out postreplication repair involves translesion synthesis (TLS) past damaged sites. TLS is de®cient in the variant form of the sun-sensitive cancer-prone disorder xeroderma pigmentosum (XP). The gene defective in these XP variants (XP-V) encodes a DNA polymerase, polh (Johnson et al., 1999;Masutani et al., 1999), which is able to replicate undamaged templates or those containing cyclobutane pyrimidine dimers (CPDs, the major UV photoproduct) with equal ef®ciencies (Masutani et al., 1999). TLS by polh is the principal mechanism for bypassing CPDs in human cells. Although the lack of polh in XP-V cells does not confer substantial hypersensitivity to killing by UV light, UV hypermutability is increased to levels approaching those in classical XP cells, which are de®cient in nucleotide excision repair (Maher et al., 1976).Polh is a member of the recently discovered Y-family of DNA polymerases (Ohmori et al., 2001), which have been best characterized for their lesion-bypassing properties (reviewed in Goodman, 2002). There are, however, few studies to date to indicate how these polymerases function inside cells. In previous work, we showed that in S-phase cells, polh localizes in replication foci. On exposure to DNA-damaging treatments, we observed an accumulation of polh-containing foci. These appear to represent replication factories in which replication forks are stalled at lesions (Kannouche et al., 2001). The C-terminal 70 amino acids of polh are requ...

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPKdependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Microcephalin (MCPH1/BRIT1) forms ionizing radiationinduced nuclear foci (IRIF) and is required for DNA damage-responsive S and G 2 -M-phase checkpoints. MCPH1 contains three BRCT domains. Here we report the cloning of chicken Mcph1 (cMcph1) and functional analysis of its individual BRCT domains. Full-length cMcph1 localized to centrosomes throughout the cell cycle and formed IRIF that colocalized with c-H2AX. The tandem C-terminal BRCT2 and BRCT3 domains of cMcph1 were necessary for IRIF formation, while the N-terminal BRCT1 was required for centrosomal localization in irradiated cells. Centrosomal targeting of cMcph1 was independent of ATM, Brca1 or Chk1. cMcph1 formed IRIF in ATM-and Brca1-deficient cells, but not in H2AX-deficient cells. Inability to form cMcph1 IRIF impaired the cellular response to DNA damage. These results suggest that the role of microcephalin in the vertebrate DNA damage response is controlled by interaction of the Cterminal BRCT domains with c-H2AX. The tumour suppressor BRCA1 is involved in numerous activities that maintain genome stability (Starita and Parvin, 2003). Crucial to the functions of BRCA1 are a pair of C-terminal domains, which can recognize phosphopeptides and mediate protein-protein interactions in the response to DNA damage (Manke et al., 2003;Yu et al., 2003). These BRCA1 C-terminal (BRCT) repeats are found in a large superfamily of proteins involved in cellular responses to genotoxic stress (Glover et al., 2004).One member of this BRCT domain-containing superfamily is microcephalin (MCPH1). MCPH1 is encoded by MCPH1/BRIT1 (hereafter MCPH1), a gene mutated in primary microcephaly (OMIM 251200) (Jack- (Rogakou et al., 1999). A recent study (Rai et al., 2006) found that depletion of MCPH1 in human cells blocked formation of IRIF by NBS1, 53BP1, phosphorylated ATM and MDC1, but not g-H2AX. DNA damage-induced chromatin association of several DNA damage response proteins was also blocked by MCPH1 RNAi and a high level of chromosome aberrations was observed. MCPH1 aberrations were frequently detected in breast, ovarian and prostate cancer (Rai et al., 2006). Together, these data suggest an important role for MCPH1 in DNA damage responses and in preventing cellular transformation.We identified the chicken MCPH1 orthologue by database analysis, cloned it by RT-PCR and confirmed the sequence (DDBJ/EMBL/GenBank accession no. DQ788861). Zebrafish Mcph1 sequence was derived by database analysis and these sequences were aligned with known mammalian Mcph1 sequences using ClustalW. As shown in Supplementary Figure S1, all vertebrate sequences analysed showed similar organization, with a highly conserved single N-terminal BRCT domain and a pair of C-terminal BRCT domains, which will be referred to here as BRCT1, BRCT2 and BRCT3, respectively. The conserved BRCT1 and BRCT2-BRCT3 regions are separated by a poorly conserved region that does not contain any distinct structural motifs, as determined by SMART domain search at http://smart.embl-heidelberg.de/. These findings suggest that k...

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

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Winchester syndrome (WS, MIM #277950) is an extremely rare autosomal recessive skeletal dysplasia characterized by progressive joint destruction and osteolysis. To date, only one missense mutation in MMP14, encoding the membrane-bound matrix metalloprotease 14, has been reported in WS patients. Here, we report a novel hypomorphic MMP14 p.Arg111His (R111H) allele, associated with a mitigated form of WS. Functional analysis demonstrated that this mutation, in contrast to previously reported human and murine MMP14 mutations, does not affect MMP14's transport to the cell membrane. Instead, it partially impairs MMP14's proteolytic activity. This residual activity likely accounts for the mitigated phenotype observed in our patients. Based on our observations as well as previously published data, we hypothesize that MMP14's catalytic activity is the prime determinant of disease severity. Given the limitations of our in vitro assays in addressing the consequences of MMP14 dysfunction, we generated a novel mmp14a/b knockout zebrafish model. The fish accurately reflected key aspects of the WS phenotype including craniofacial malformations, kyphosis, short-stature and reduced bone density due to defective collagen remodeling. Notably, the zebrafish model will be a valuable tool for developing novel therapeutic approaches to a devastating bone disorder.