RecQ-like helicases, which include 5 members in the human genome, are important in maintaining genome integrity. We present a crystal structure of a truncated form of the human RECQ1 protein with Mg-ADP. The truncated protein is active in DNA fork unwinding but lacks other activities of the full-length enzyme: disruption of Holliday junctions and DNA strand annealing. The structure of human RECQ1 resembles that of Escherichia coli RecQ, with some important differences. All structural domains are conserved, including the 2 RecA-like domains and the RecQ-specific zinc-binding and winged-helix (WH) domains. However, the WH domain is positioned at a different orientation from that of the E. coli enzyme. We identify a prominent ␤-hairpin of the WH domain as essential for DNA strand separation, which may be analogous to DNA strand-separation features of other DNA helicases. This hairpin is significantly shorter in the E. coli enzyme and is not required for its helicase activity, suggesting that there are significant differences between the modes of action of RecQ family members.DNA helicase ͉ DNA repair ͉ Holliday junction ͉ structural genomics ͉ winged helix T he RecQ helicases are a family of DNA-unwinding enzymes conserved from prokaryotes to mammals that play a key role in the maintenance of genome stability. The RecQ helicase family has 5 representatives in the human genome (1-3): RECQ1 (also known as RECQL or RECQL1), BLM, WRN, RECQ4, and RECQ5. Although these 5 enzymes are similar in their catalytic core, they probably have distinct functions, as indicated by the genetic disorders associated to mutations in the genes of BLM, WRN, and RECQ4. In particular, mutations in the gene encoding for BLM (4) are associated with the Bloom's syndrome (BS), which is manifested as an increased incidence of a wide spectrum of cancers. Werner's syndrome (WS), which is linked to mutations in the WRN (5) gene, involves many signs of premature aging, as well as a predisposition to a more limited spectrum of cancers. Mutations in the gene of RECQ4 are the cause of more varied genetic disease phenotypes, including Rothmund-Thomson (RTS) (6, 7), RAPADILINO (8), and Baller-Gerold (9) syndromes. No disease phenotypes have been associated with mutations in the genes of the other 2 family members, RECQ1 and RECQ5 yet, although they may be responsible for additional cancer predisposition disorders that are distinct from RTS, BS, and WS. In this regard, interesting candidates are patients with a phenotype similar to that of RTS individuals who do not carry any mutations in the RECQ4 gene (7). A possible role of RECQ1 in genome maintenance is suggested by several observations (reviewed in ref 10). Biochemical purification from human embryonic kidney cells recovered RECQ1 as the major Holliday junction (HJ) branch migration activity (11). Knockout of the RECQ1 gene in mice (12) or suppression of its expression in HeLa cells (11) resulted in cellular phenotypes that include chromosomal instability, increased sister chromatid exchange, and hei...

The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 Å crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-HisAsp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the 0 and +1 subsites by (GlcNAc) 2 for activity and proceeds through a tetrahedral oxyanion intermediate.Colletotrichum lindemuthianum is a plant fungal pathogen found extensively in tropical and subtropical regions. Colletotrichum species are the causative agent of anthracnose that affects economically important crop species (1). Furthermore, Colletotrichum sp. have recently been reported to cause subcutaneous and systemic infections among immunosuppressed patients (2). Colletotrichum sp. are facultative biotrophs. Before the fungal hyphae can successfully penetrate and gain access to host tissue, the fungus has to first evade plant antimicrobial hydrolases such as chitinases and -(1,3)glucanases (3, 4). The chitinases degrade fungal chitin, an insoluble linear polymer of -(1-4)-linked N-acetylglucosamine (GlcNAc). 1 The breakdown products may act as elicitors of active defense responses within the plant (5-7). Studies of cell wall composition of invasive fungal hyphae suggest that exposed fungal chitin polymers are partially de-N-acetylated during infection and initial growth within the host (8). Chitosan, the de-N-acetylated product, is a poor substrate for chitinases, which require the presence of N-acetyl moieties for recognition and catalysis (9). Thus, conversion of chitin to chitosan during plant extracellular colonization may protect pathogenic fungal hyphae from being lysed by secreted plant chitinases. The enzyme responsible for chitin modification is a developmentally regulated, secreted, chitin deacetylase (CDA) (10). C. lindemuthianum chitin deacetylase (ClCDA) is a member of the family 4 carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/∼cazy/CAZY (11)], which include several members that share the primary structure assigned as the "NodB homology domain" (12). Rh...