Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity. As a result, each superfamily is subdivided, where appropriate, on the basis of mechanistic understanding, which we hope will provide a framework for classification of new superfamily members as they are discovered and characterized.

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.

We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.

The stalling of DNA replication forks that occurs as a consequence of encountering DNA damage is a critical problem for cells. RecG protein is involved in the processing of stalled replication forks, and acts by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass. We have determined the crystal structure of RecG bound to a DNA substrate that mimics a stalled replication fork. The structure not only reveals the elegant mechanism used by the protein to recognize junctions but has also trapped the protein in the initial stage of fork reversal. We propose a mechanism for how forks are processed by RecG to facilitate replication fork restart. In addition, this structure suggests that the mechanism and function of the two largest helicase superfamilies are distinct.

In eukaryotic cells, RAD52 protein plays a central role in genetic recombination and DNA repair by (i) promoting the annealing of complementary single-stranded DNA and (ii) stimulation of the RAD51 recombinase. The single-strand annealing domain resides in the N-terminal region of the protein and is highly conserved, whereas the nonconserved RAD51-interaction domain is located in the C-terminal region. An N-terminal fragment of human RAD52 (residues 1-209) has been purified to homogeneity and, similar to the full-size protein (residues 1-418), shown to promote singlestrand annealing in vitro. We have determined the crystal structure of this single-strand annealing domain at 2.7 Å. The structure reveals an undecameric (11) subunit ring with extensive subunit contacts. A large, positively charged groove runs along the surface of the ring, readily suggesting a mechanism by which RAD52 presents the single strand for reannealing with complementary single-stranded DNA.recombination ͉ DNA repair ͉ crystallography T he ability of a cell to survive agents that promote genome breakage requires efficient recombinational repair systems. In lower eukaryotes, repair involves the RAD52 epistasis group of genes including RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2 (1,2). A key member of this group, RAD52, encodes a protein that plays a dual role in recombination by (i) promoting the annealing of complementary single strands (3-5) and (ii) stimulating in vitro recombination reactions catalyzed by the RAD51 recombinase (6-8).Human RAD52 protein shares many properties with its yeast counterpart including DNA strand annealing and RAD51 stimulation activities (9-11). The human protein has been visualized by electron microscopy, and a low-resolution three-dimensional structure has been determined (12, 13). Human RAD52 consists of seven subunits that are organized in the form of a ring with a large central channel. The Saccharomyces cerevisiae RAD52 protein has been shown also to form rings, but no detailed structure is available presently (4).The mechanism by which RAD52 promotes single-stranded DNA (ssDNA) annealing is unknown. However, when complexes formed between RAD52 and ssDNA were probed with hydroxyl radicals, a unique repeating four-nucleotide hypersensitivity pattern was observed (14). Sequence-independent hypersensitivity was observed over Ϸ36 nucleotides and was phased precisely from the terminal nucleotide. These results led to the proposal that RAD52 binds ssDNA via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein ring.Sequence comparisons, site-directed mutagenesis, and biochemical studies indicate that the highly conserved N-terminal region of RAD52 possesses ssDNA annealing activities, whereas the RAD51-interaction domain is located toward the nonconserved C-terminal region (15-18). Furthermore, the RAD59 protein shares sequence homology with the N-terminal region of RAD5...