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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumDomain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNAXiong Yu*, Steven A.Jacobs*, Stephen C.West†, Tomoko Ogawa‡, and Edward H.Egelman*§ *Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Box 800733, Charlottesville, VA 22908; †Clare Hall Laboratories, Imperial Cancer Research Fund, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, United Kingdom; and ‡National Institute of Genetics, Yata, Mishima, Shizuoka 411–8540, Japan Both the bacterial RecA protein and the eukaryotic Rad51 protein form helical nucleoprotein filaments on DNA that catalyze strand transfer between two homologous DNA molecules. However, only the ATP-binding cores of these proteins have been conserved, and this same core is also found within helicases and the F1-ATPase. The C-terminal domain of the RecA protein forms lobes within the helical RecA filament. However, the Rad51 proteins do not have the C-terminal domain found in RecA, but have an N-terminal extension that is absent in the RecA protein. Both the RecA C-terminal domain and the Rad51 N-terminal domain bind DNA. We have used electron microscopy to show that the lobes of the yeast and human Rad51 filaments appear to be formed by N-terminal domains. These lobes are conformationally flexible in both RecA and Rad51. Within RecA filaments, the change between the “active” and “inactive” states appears to mainly involve a large movement of the C-terminal lobe. The N-terminal domain of Rad51 and the C-terminal domain of RecA may have arisen from convergent evolution to play similar roles in the filaments. The Escherichia coli RecA protein has served as a model for understanding protein-mediated genetic recombination (1, 2). RecA plays an important role in DNA repair, and studies of RecA continue to provide insight into how repair, replication, and recombination functions are intimately linked. RecA homologs, such as RadA, UvsX, Dmc1, and Rad51, have now been identified in many organisms. Evidence in support of a key role of Rad51 in recombination, repair (3, 4) and cancer (5) in humans has emerged over the past several years. Although RecA is not an essential gene in E. coli, it has been shown that RAD51 knockouts are lethal in both chicken and mammalian cell lines (6–8). Chromosome fragmentation occurs after RAD51 inactivation in chicken DT40 cells, showing that RAD51 is required for the repair of stalled or broken replication forks in proliferating cells (8). Alignments of the RecA and Rad51 protein sequences (9, 10) have shown that, outside of the homologous core (containing the nucleotide binding site), RecA has a C-terminal extension that is absent in Rad51 and that the Rad51 proteins have an N-terminal extension that is absent in RecA. The Saccharomyces cerevisiae Rad51 (ScRad51) N-terminal extension is even longer than that found in the human protein (hRad51). The homologous core structure has also been found in the F1-ATPase (11) and in several helicases (12–15), suggesting that all of these proteins have diverged from a common ancestor. Although there is no apparent homology between the N-terminal domain of Rad51 and the C-terminal domain of RecA, it has been reported that the C-terminal domain of RecA binds double-stranded DNA (dsDNA) (16, 17) and that the N-terminal domain of hRad51 binds both single-stranded DNA (ssDNA) and dsDNA (18). The active state of RecA appears to be a nucleoprotein filament formed on DNA (19, 20). The T4 UvsX protein (21), the ScRad51 protein (22), and the hRad51 protein (23) induce the same unusual conformation in DNA as that induced by the RecA protein: ≈5.1 Å rise per base pair (from 3.4 Å in B-DNA) and ≈18.6 bp per turn (from 10.5 in B-DNA). This extended filament is found with RecA bound to either ssDNA or dsDNA. In contrast, the extended filaments have been seen only with hRad51 filaments formed on dsDNA, whereas filaments formed on ssDNA were relatively compressed (23). We show in this paper that, under the appropriate conditions, extended hRad51 filaments can also be seen on ssDNA. Thus, RecA, UvsX, and Rad51, although they have relatively weak overall sequence similarity, change the pitch of DNA from ≈36 Å to ≈95 Å. It has been suggested that this unusual DNA conformation has been the basis for the conservation of these nucleoprotein filaments from bacteria to humans (22). We have used a new approach to image analysis for looking at the filaments formed by RecA and Rad51. This approach has not only provided us with more detail, but has allowed us to visualize multiple conformational states of these filaments. We have been able to interpret the differences between these conformational states in terms of the domain structure of these proteins. MethodsPreparation of RecA-DNA and hRad51-DNA Complexes. The RecA protein was purified as described (24). Circular Electron Microscopy. Samples were applied to carbon-coated grids and negatively stained with 1% uranyl acetate. Specimens were This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.
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X174 dsDNA (GIBCO/BRL) was linearized (
were formed by incubating 6 µM hRad51 in 25 mM triethanolamine-HCl (Fisher) buffer (pH 7.2) at 37°C for 5 min, with M13 ssDNA and 2.5 mM ATP (Sigma). The ssDNA was present at a Rad51:ssDNA ratio of 80:1 (wt/wt). Then NaF (Aldrich) and Al(NO3)2 (Aldrich) were added to a final concentration of 2.5 mM, and the reaction mixture was incubated at 37°C for an additional 15 min.