RESUMO
DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA , RNA Polimerase II , RNA , Humanos , RNA/metabolismo , RNA/genética , RNA/química , DNA/metabolismo , DNA/química , RNA Polimerase II/metabolismo , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/genética , RNA Helicases/metabolismo , RNA Helicases/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Estruturas R-Loop , Fosforilação , Homeostase/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
BACKGROUND: Proper DNA replication is essential for faithful transmission of the genome. However, replication stress has serious impact on the integrity of the cell, leading to stalling or collapse of replication forks, and has been determined as a driving force of carcinogenesis. Mus81-Mms4 complex is a structure-specific endonuclease previously shown to be involved in processing of aberrant replication intermediates and promotes POLD3-dependent DNA synthesis via break-induced replication. However, how replication components might be involved in this process is not known. RESULTS: Herein, we show the interaction and robust stimulation of Mus81-Mms4 nuclease activity by heteropentameric replication factor C (RFC) complex, the processivity factor of replicative DNA polymerases that is responsible for loading of proliferating cell nuclear antigen (PCNA) during DNA replication and repair. This stimulation is enhanced by RFC-dependent ATP hydrolysis and by PCNA loading on the DNA. Moreover, this stimulation is not specific to Rfc1, the largest of subunit of this complex, thus indicating that alternative clamp loaders may also play a role in the stimulation. We also observed a targeting of Mus81 by RFC to the nick-containing DNA substrate and we provide further evidence that indicates cooperation between Mus81 and the RFC complex in the repair of DNA lesions generated by various DNA-damaging agents. CONCLUSIONS: Identification of new interacting partners and modulators of Mus81-Mms4 nuclease, RFC, and PCNA imply the cooperation of these factors in resolution of stalled replication forks and branched DNA structures emanating from the restarted replication forks under conditions of replication stress.
Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteína de Replicação C/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Recombinação Genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations.
Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Replicação do DNA , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , DNA Helicases/genética , Instabilidade Genômica , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Domínios ProteicosRESUMO
Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Reparo de DNA por Recombinação , Motivos de Aminoácidos , DNA/biossíntese , DNA Polimerase III/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases/fisiologia , Raios UltravioletaRESUMO
Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer-template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3'-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA.
Assuntos
Dano ao DNA , Replicação do DNA , Fatores de Transcrição Forkhead/metabolismo , Adenosina Trifosfatases/metabolismo , DNA/química , DNA/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Humanos , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Moldes GenéticosRESUMO
Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.
Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Pareamento Cromossômico , Sequência Conservada , DNA/metabolismo , Dano ao DNA , Análise Mutacional de DNA , Primers do DNA/metabolismo , Instabilidade Genômica , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.