The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas prebinding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.DNA helicases unwind duplex DNA and are essential components of the DNA replication, recombination, and repair machinery in all cellular organisms and many viruses. DNA helicases utilize the energy released by ATP hydrolysis to undergo conformational cycling and translocate along singlestranded DNA (ssDNA), 4 displacing a duplex DNA strand in the process. Many helicases belong to one of three superfamilies (SF1, 2, and 3), classified according to the conservation of specific sequence motifs (1). SF1 and SF2 helicases possess two motor domains with RecA-like folds that couple ATP hydrolysis to DNA translocation (2). SF2 DNA helicases include RecG in bacteria, hepatitis C virus NS3, and the RecQ family helicases, which all translocate along ssDNA with a 3Ј to 5Ј polarity (3). The RecQ helicases play a key role in maintaining genomic integrity by stabilizing stalled replication forks and removing intermediates of DNA recombination (4). Previous studies have shown that RecQ proteins target specialized DNA structures, specifically branched substrates that mimic replication forks and Holliday junctions. In humans, RecQ family helicases include the BLM and WRN proteins, mutated in certain rare inherited diseases in humans (5). The Hel308 family SF2 helicases, like RecQ, are implicated in DNA repair, recombination, and genome stability. The founding member, Mus308 from Drosophila melanogaster, was identified in a screen for mutations conferring hypersensitivity to DNA cross-linking reagents (6). Mus308 consists of an N-terminal SF2 helicase fused to a C-terminal DNA polymerase. The human ortholog, PolQ, has the same arrangement (7), and the polymerase domain has been sho...

PDB Reference: PCNA, 2ix2, r2ix2sf.PCNA is a ring-shaped protein that encircles DNA, providing a platform for the association of a wide variety of DNA-processing enzymes that utilize the PCNA sliding clamp to maintain proximity to their DNA substrates. PCNA is a homotrimer in eukaryotes, but a heterotrimer in crenarchaea such as Sulfolobus solfataricus. The three proteins are SsoPCNA1 (249 residues), SsoPCNA2 (245 residues) and SsoPCNA3 (259 residues). The heterotrimeric protein crystallizes in space group P2 1 , with unit-cell parameters a = 44.8, b = 78.8, c = 125.6 Å , = 100.5 . The crystal structure of this heterotrimeric PCNA molecule has been solved using molecular replacement. The resulting structure to 2.3 Å sheds light on the differential stabilities of the interactions observed between the three subunits and the specificity of individual subunits for partner proteins.