Ultraviolet (UV) light-induced pyrimidine photodimers are repaired by the nucleotide excision repair pathway. Photolesions have biophysical parameters closely resembling undamaged DNA, impeding discovery through damage surveillance proteins. The DDB1-DDB2 complex serves in the initial detection of UV lesions in vivo. Here we present the structures of the DDB1-DDB2 complex alone and bound to DNA containing either a 6-4 pyrimidine-pyrimidone photodimer (6-4PP) lesion or an abasic site. The structure shows that the lesion is held exclusively by the WD40 domain of DDB2. A DDB2 hairpin inserts into the minor groove, extrudes the photodimer into a binding pocket, and kinks the duplex by approximately 40 degrees. The tightly localized probing of the photolesions, combined with proofreading in the photodimer pocket, enables DDB2 to detect lesions refractory to detection by other damage surveillance proteins. The structure provides insights into damage recognition in chromatin and suggests a mechanism by which the DDB1-associated CUL4 ubiquitin ligase targets proteins surrounding the site of damage.

SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.

Believed to have been critical to the origin of life on Earth 1, the hydrosulfide ion (HS−) and its undissociated form, hydrogen sulfide (H2S), continue to play a prominent role in physiology and cellular signaling 2. As a major metabolite in anaerobic bacterial growth, hydrogen sulfide is a product of both assimilatory and dissimilatory sulfate reduction 2–4. These pathways can reduce various oxidized sulfur compounds including sulfate, sulfite and thiosulfate. The dissimilatory sulfate reduction pathway uses this molecule as the terminal electron acceptor for anaerobic respiration, where it produces excess amounts of H2S4. The reduction of sulfite is a key intermediate step in all sulfate reduction pathways. In Clostridium and Salmonella, an inducible sulfite reductase is directly linked to the regeneration of NAD+, which has been suggested to play a role in energy production and growth, as well as in the detoxification of sulfite 3. Above a certain concentration threshold, both H2S and HS− nhibit cell growth by binding the metal centers of enzymes and cytochrome oxidase5, necessitating a release mechanism for the export of this toxic metabolite from the cell 5–9. Through a combination of genetic, biochemical and functional approaches, we have identified a hydrosulfide ion channel (HSC) in the pathogen Clostridium difficile. The HS− channel is a member of the formate-nitrite-transport (FNT) family, in which ~50 HSC genes form a third subfamily alongside those for formate (FocA) 10,11 and for nitrite (NirC) 12. In addition to HS− ions, HSC is also permeable to formate and nitrite. Such polyspecificity can be explained by the conserved ion selectivity filter observed in the HSC crystal structure. The channel has a low open probability and is tightly regulated, to avoid decoupling of the membrane proton gradient.

Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WS gene (WRN) and a deficiency in the function of the Werner protein (WRN).WRN is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3-5-exonuclease, an ATPase, and a 3-5-helicase. Deficiency in WRN results in a cellular phenotype of genomic instability. The biochemical characteristics of WRN and the cellular phenotype of WRN mutants suggest that WRN plays an important role in DNA metabolic pathways such as recombination, transcription, replication, and repair. The catalytic activities of WRN have been extensively studied and are fairly well understood. However, much less is known about the domain-specific interactions between WRN and its DNA substrates. This study identifies and characterizes three distinct WRN DNA binding domains using recombinant truncated fragments of WRN and five DNA substrates (long forked duplex, blunt-ended duplex, single-stranded DNA, 5-overhang duplex, and Holliday junction). Substrate-specific DNA binding activity was detected in three domains, one Nterminal and two different C-terminal WRN fragments (RecQ conserved domain and helicase RNase D conserved domain-containing domains). The substrate specificity of each DNA binding domain may indicate that each protein domain has a distinct biological function. The importance of these results is discussed with respect to proposed roles for WRN in distinct DNA metabolic pathways.Werner syndrome (WS) 1 is a rare autosomal recessive disorder characterized by the early onset of aging symptoms, such as graying of the hair, cataracts, osteoporosis, atherosclerosis, type II diabetes mellitus, and high incidence of malignant neoplasm (1). The gene mutated in WS (WRN), encodes a nuclear 1432-amino acid protein (WRN) (2). WRN belongs to the RecQ helicase family of proteins, which is a conserved group of proteins implicated in several aspects of DNA metabolism (2, 3). Three of the RecQ helicases, WRN, Bloom (BLM), and Rothmund Thomsons, are associated with heritable human diseases (3). WRN is a multifunctional protein with three DNA-dependent catalytic activities: 3Ј-5Ј-exonuclease (4, 5), ATPase, and 3Ј-5Ј-helicase (6) (Fig. 1A). WRN is the only member of the human RecQ family known to include an exonuclease domain.Cells from WS patients display replication defects, genomic instability, and altered telomere dynamics, suggesting an important role for WRN in DNA metabolic pathways (for review, see Ref. 7). This is consistent with the observation that many proteins that interact with WRN are involved in recombination, replication, transcription, and telomere structure or repair (8 -15). In vitro, WRN binds and is catalytically active toward non-canonical DNA structures such as recombination intermediates (16), replication forks (17), repair intermediates (13, 18), and telomeric ends (19). For example, WRN interacts with Holliday junctions, forked duplexes, 5Ј-overhang duplexes, and D-loops.Typically, WS cells carry a C-terminal truncated WRN that...