RESUMO
DNA interstrand crosslinks (ICLs), highly toxic lesions that covalently link the Watson and Crick strands of the double helix, are repaired by a complex, replication-coupled pathway in higher eukaryotes. The earliest DNA processing event in ICL repair is the incision of parental DNA on either side of the ICL ("unhooking"), which allows lesion bypass. Incisions depend critically on the Fanconi anemia pathway, whose activation involves ubiquitylation of the FANCD2 protein. Using Xenopus egg extracts, which support replication-coupled ICL repair, we show that the 3' flap endonuclease XPF-ERCC1 cooperates with SLX4/FANCP to carry out the unhooking incisions. Efficient recruitment of XPF-ERCC1 and SLX4 to the ICL depends on FANCD2 and its ubiquitylation. These data help define the molecular mechanism by which the Fanconi anemia pathway promotes a key event in replication-coupled ICL repair.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Recombinases/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Clivagem do DNA , Dano ao DNA , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases , Endonucleases/química , Exodesoxirribonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Humanos , Cinética , Enzimas Multifuncionais , Ligação Proteica , Recombinases/química , Ubiquitinação , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
XPF-ERCC1 is a structure-specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease-specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of XPF-ERCC1 in DNA interstrand crosslink (ICL) repair. We used Xenopus egg extracts to measure both ICL and nucleotide excision repair, and we identified mutations that are specifically defective in ICL repair. One of these separation-of-function mutations resides in the helicase-like domain of XPF and disrupts binding to SLX4 and recruitment to the ICL A small deletion in the same domain supports recruitment of XPF to the ICL, but inhibited the unhooking incisions most likely by disrupting a second, transient interaction with SLX4. Finally, mutation of residues in the nuclease domain did not affect localization of XPF-ERCC1 to the ICL but did prevent incisions on the ICL substrate. Our data support a model in which the ICL repair-specific function of XPF-ERCC1 is dependent on recruitment, positioning and substrate recognition.
Assuntos
Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Animais , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , XenopusRESUMO
DNA repair pathways are crucial to maintain the integrity of our genome and prevent genetic diseases such as cancer. There are many different types of DNA damage and specific DNA repair mechanisms have evolved to deal with these lesions. In addition to these repair pathways there is an extensive signaling network that regulates processes important for repair, such as cell cycle control and transcription. Despite extensive research, DNA damage repair and signaling are not fully understood. In vitro systems such as the Xenopus egg extract system, have played, and still play, an important role in deciphering the molecular details of these processes. Xenopus laevis egg extracts contain all factors required to efficiently perform DNA repair outside a cell, using mechanisms conserved in humans. These extracts have been used to study several genome maintenance pathways, including mismatch repair, non-homologous end joining, ICL repair, DNA damage checkpoint activation, and replication fork stability. Here we describe how the Xenopus egg extract system, in combination with specifically designed DNA templates, contributed to our detailed understanding of these pathways.
Assuntos
Dano ao DNA , Óvulo/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animais , Fracionamento Celular , Reparo do DNA , Replicação do DNA , Feminino , Genoma , Modelos Genéticos , Transdução de SinaisRESUMO
The invasion-associated type III secretion system (T3SS-1) of Salmonella enterica serotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasion in vitro but required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responses in vitro and in vivo.