RESUMO
RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by â¼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.
Assuntos
DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , Proteínas Motores Moleculares/metabolismo , RecQ Helicases/metabolismo , Imagem Individual de Molécula , Trifosfato de Adenosina/metabolismo , DNA de Cadeia Simples/ultraestrutura , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Proteínas Motores Moleculares/ultraestrutura , Mutação de Sentido Incorreto , Mutação Puntual , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , RecQ Helicases/ultraestrutura , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/metabolismo , Especificidade por SubstratoRESUMO
RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo-EM structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read-through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II-TFIIS interaction.
Assuntos
Mimetismo Molecular , RNA Polimerase II/química , RecQ Helicases/química , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , RNA Polimerase II/metabolismo , RNA Polimerase II/ultraestrutura , RecQ Helicases/metabolismo , RecQ Helicases/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/fisiologia , Fatores de Elongação da Transcrição/ultraestruturaRESUMO
BACKGROUND: Pyrococcus furiosus Hjm (PfuHjm) is a structure-specific DNA helicase that was originally identified by in vitro screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Theta (PolTheta), HEL308, and Drosophila Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that PfuHjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by PfuHjm will require its three-dimensional structure at atomic resolution. RESULTS: We determined the crystal structures of PfuHjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0-2.7 A. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of Archaeoglobus fulgidus Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolTheta, indicating the strong conformational conservation between archaea and eukarya. CONCLUSION: The detailed comparison between our DNA-free PfuHjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.