Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.The hereditary syndrome Fanconi anemia (FA) belongs to a group of caretaker gene diseases characterized by genomic instability and increased susceptibility to cancer. A hallmark of FA is cellular hypersensitivity to DNA interstrand cross-links (ICLs), suggesting a defect in the DNA damage response (18,19,48). Twelve FA complementation groups have been identified, and the majority of the corresponding genes have been cloned (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCJ, FANCL, and FANCM) (22,23,25,43,55,59,60,66,87,93). Although the function of the FA proteins is unknown, identification of BRCA2 (breast cancer-associated gene 2) as FANCD1 and of FANCJ as the BRCA1-associated helicase gene Brip1/BACH1, suggests convergence of the FA/BRCA pathway with a larger network of proteins involved in DNA repair (7,(51)(52)(53)95). This is underscored by the discovery that FANCM is related to archaeal Hef, a protein that binds and processes irregular arrangements of DNA in branched structures resembling replication forks (50,71,72).According to current models, the FA pathway consists of an upstream nuclear core complex, including FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, required for the activation of its target, FANCD2 (24, 34-36, 59, 60, 66). FANCD2 is monoubiquitinated during S phase and in response to various types of DNA damage, including DNA ICLs, DNA double-strand breaks (DSBs), and replication fork stalling (36, 89). DNA damage-induced monoubiquitination of FANCD2 is also reduced in cells from Seckel syndrome patients with a defect in the ataxia telangiectasia-and RAD3-related gene, ATR (1), suggesting that the FA pathway is under at least partial control of the ATR kinase. Monoubiquitination of FANCD2 is required for its association with c...

Although it is well established that DNA-protein crosslinks are formed as a consequence of cellular exposure to agents such as formaldehyde, transplatin, ionizing and ultraviolet radiation, the biochemical pathways that promote cellular survival via repair or tolerance of these lesions are poorly understood. To investigate the mechanisms that function to limit DNA-protein crosslink-induced cytotoxicity, the Saccharomyces cerevisiae non-essential gene deletion library was screened for increased sensitivity to formaldehyde exposure. Following low-dose, chronic exposure, strains containing deletions in genes mediating homologous recombination showed the greatest sensitivity, while under the same exposure conditions, deletions in genes associated with nucleotide excision repair conferred only low to moderate sensitivities. However, when the exposure regime was changed to a high-dose acute (short-term) formaldehyde treatment, the genes that conferred maximal survival switched to the nucleotide excision repair pathway, with little contribution of the homologous recombination genes. Data are presented which suggest that following acute formaldehyde exposure, repair and/or tolerance of DNA-protein crosslinks proceeds via formation of nucleotide excision repair-dependent single-strand break intermediates and without a detectable accumulation of doublestrand breaks. These data clearly demonstrate a differential pathway response to chronic versus acute formaldehyde exposures and may have significance and implications for risk extrapolation in human exposure studies.

In this study, we collected water from different locations in 32 drinking water distribution networks in the Netherlands and analysed the spatial and temporal variation in microbial community composition by high-throughput sequencing of 16S rRNA gene amplicons. We observed that microbial community compositions of raw source and processed water were very different for each distribution network sampled. In each network, major differences in community compositions were observed between raw and processed water, although community structures of processed water did not differ substantially from end-point tap water. End-point water samples within the same distribution network revealed very similar community structures. Network-specific communities were shown to be surprisingly stable in time. Biofilm communities sampled from domestic water metres varied distinctly between households and showed no resemblance to planktonic communities within the same distribution networks. Our findings demonstrate that high-throughput sequencing provides a powerful and sensitive tool to probe microbial community composition in drinking water distribution systems. Furthermore, this approach can be used to quantitatively compare the microbial communities to match end-point water samples to specific distribution networks. Insight in the ecology of drinking water distribution systems will facilitate the development of effective control strategies that will ensure safe and high-quality drinking water.

In addition to monoubiquitination by the FA core complex, FANCD2 and FANCI are phosphorylated by the two major cell cycle checkpoint kinases, ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related),y in response to DNA damage (2-6). ATM-dependent phosphorylation of FANCD2 occurs following ionizing irradiation and is required for activation of the ionizing irradiation-induced intra-S phase checkpoint (4). ATR-dependent phosphorylation of FANCD2 is triggered by various types of DNA damage, including replication stress, and is required for the interstrand cross-link-induced intra-S phase checkpoint response (2, 3). Moreover, phosphorylation by ATR is required for efficient FANCD2 monoubiquitination in response to DNA damage, suggesting that the FA pathway might participate in ATR-dependent coordination of the S phase of the cell cycle (3, 7).The recent identification of a highly conserved FA core complex member, FANCM (8, 9), indicates a direct role of FA pathway proteins in repair steps at sites of DNA damage. FANCM is a homolog of the archaebacterial Hef protein (helicase-associated endonuclease for fork-structured DNA) and contains two DNA processing domains: a DEAH box helicase domain and an XPF/ERCC4-like endonuclease domain. FANCM has ATP-dependent DNA translocase activity and can dissociate DNA triple helices in vitro (8). Moreover, FANCM binds Holliday junctions and DNA replication fork structures in vitro and promotes ATP-dependent branch point migration, suggesting that FANCM might be involved in DNA processing at stalled replication forks (10,11). In human cells, FANCM localizes to chromatin and is required for chromatin recruitment of other FA core complex proteins (8,12). FANCM is phosphorylated during both the M and S phases and in response to DNA-damaging agents (8,12,13). Interestingly, DNA damage-induced phosphorylation of FANCM is independent of the FA core complex (8), suggesting that FANCM is controlled by other, as yet unknown upstream components of the DNA damage response. Here, we used cell-free Xenopus egg extracts to investigate the role of FANCM during replication and in the DNA damage response. We show that Xenopus FANCM (xFANCM) binds chromatin in a replication-dependent manner and is phosphorylated during unperturbed replication as well as in response to various DNA damage structures. Both chromatin recruitment and phosphorylation of xFANCM are partially controlled by xFANCD2, suggesting feedback signaling from xFANCD2 to the upstream xFA core complex via regulation of xFANCM. In addition, chromatin recruitment during unperturbed replication and activation of xFANCM in response to DNA damage are controlled by the xATR and xATM cell cycle kinases.

In 4 of 17 Helicobacter pylori strains obtained from antral biopsy samples, the registered primary resistance (MIC, >32 g/ml) appeared to be nonstable after prolonged microaerophilic incubation. In all resistant strains tested, susceptibility could be obtained when culture under normal microaerophilic conditions was preceded by a period of anaerobic incubation. Both of these findings may explain the observed discrepancy between the results of in vitro susceptibility tests and the eradication obtained in vivo.