RESUMO
Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.
Assuntos
Dano ao DNA , Reparo do DNA , DNA/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , DNA/genética , DNA/efeitos da radiação , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/ß pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5'-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Deleção de Genes , Humanos , Proteínas Monoméricas de Ligação ao GTP , Proteínas Nucleares/genética , Ligação Proteica/fisiologia , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Peptídeos/química , Proteína de Xeroderma Pigmentoso Grupo A/química , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Conformação Proteica , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
FEN-1 and XPG are members of the FEN-1 family of structure-specific nucleases, which share a conserved active site. FEN-1 plays a central role in DNA replication, whereas XPG is involved in nucleotide excision repair (NER). Both FEN-1 and XPG are active on flap structures, but only XPG cleaves bubble substrates. The spacer region of XPG is dispensable for nuclease activity on flap substrates but is required for NER activity and for efficient processing of bubble substrates. Here, we inserted the spacer region of XPG between the nuclease domains of FEN-1 to test whether this domain would be sufficient to confer XPG-like substrate specificity and NER activity on a related nuclease. The resulting FEN-1-XPG hybrid protein is active on flap and, albeit at low levels, on bubble substrates. Like FEN-1, the activity of FEN-1-XPG was stimulated by a double-flap substrate containing a 1-nt 3' flap, whereas XPG does not show this substrate preference. Although no NER activity was detected in vitro, the FEN-1-XPG hybrid displays substantial NER activity in vivo. Hence, insertion of the XPG spacer region into FEN-1 results in a hybrid protein with biochemical properties reminiscent of both nucleases, including partial NER activity.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Endonucleases Flap/química , Endonucleases Flap/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Células Cultivadas , Dano ao DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Humanos , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Raios UltravioletaRESUMO
The yeast protein Xtc1 was identified as a protein that binds directly and specifically to the activation domains of acidic activators such as E2F-1, Gal4 and VP16. Additionally, it was shown to co-purify with the RNA polymerase II holoenzyme complex and it was suggested that Xtc1 functions as a regulator of transcription that modulates the response of RNA polymerase II to transcriptional activators. We have further analyzed the transcription function of Xtc1 and show that its fusion to a heterologous DNA binding domain can repress transcription of a reporter gene in vivo in an Srb10/11-dependent manner. We suggest that the presence of Xtc1 in the RNA polymerase II holoenzyme could help to recruit an Srb10-active form of the holoenzyme to target promoters. This same protein has also been implicated in mitochondrial DNA recombination, maintenance and repair. Determination of the subcellular localization using a GFP-Xtc1 fusion shows that it localizes to both the nucleus and the mitochondria in vivo, which is consistent with Xtc1 having a function in both cellular compartments.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Nucleares , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Transcrição Gênica , Núcleo Celular/química , Núcleo Celular/metabolismo , Quinase 8 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Genes Reporter , Holoenzimas/química , Holoenzimas/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Transporte Proteico , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The retinoblastoma family of proteins are key cell cycle regulatory molecules important for the differentiation of various mammalian cell types. The retinoblastoma protein regulates transcription of a variety of genes either by blocking the activation domain of various activators or by active repression via recruitment to appropriate promoters. We show here that the retinoblastoma family of proteins functions as direct transcriptional repressors in a heterologous yeast system when fused to the DNA binding domain of Gal4. Mapping experiments indicate that either the A or the B domain of the pocket region is sufficient for repression in vivo. As is the case in mammalian cells, a phosphorylation site mutant of the retinoblastoma protein is a stronger transcriptional repressor than the wild type protein. We show that transcriptional repression by pRb is dependent on CLN3 in vivo. Furthermore, the yeast histone deacetylase components, RPD3 and SIN3, are required for transcriptional repression.