A Salmonella virulence factor activates the NOD1/NOD2 signaling pathway.

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

Making the cut : How XPF-ERCC1 unhooks DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are highly toxic lesions that bind both strands of the DNA helix together, which prevents the DNA from unwinding. This blocks important cellular processes such as DNA replication and transcription. ICL inducing agents were among the first chemotherapeutic drugs, making use of the toxic nature of ICLs especially for fast dividing cells. Today, ICL-inducing agents are still the main line of treatment for many cancers. The repair mechanisms have long been elusive, but in recent years we have begun to understand the pathways that underlie ICL repair. Much of this knowledge has come from studying Fanconi anemia (FA), a rare genetic disorder that is characterized by an extreme cellular sensitivity to ICL-inducing agents. Repair of an ICL is initiated during DNA replication when a replication fork stalls at the damaged lesion. Subsequently, the FA pathway cooperates with a variety of DNA repair proteins, such as nucleases, translesion polymerases and recombinases to coordinate ICL repair. A critical step in the repair process are the dual incisions on one of the DNA strands, to release (or ‘unhook’) the ICL, but the exact mechanism of unhooking and the nucleases directly involved in this step have remained elusive. To investigate this we have made use of the Xenopus laevis egg extract. This unique system can recapitulate the replication-dependent repair of a DNA plasmid with a single site-specific ICL. Using this cell free system we have discovered that the structure specific endonuclease XPF-ERCC1 is absolutely required for the unhooking incisions to take place. Furthermore, the recruitment of XPF-ERCC1 to the site of damage is dependent on interaction with SLX4/FANCP. Recruitment of both XPF-ERCC1 and SLX4 is in turn promoted by the ubiquitination of FANCD2, a key step in the activation of the FA pathway. We furthermore shed insight into the interaction of SLX4 and XPF, by identifying residues on XPF that are crucial for the interaction of the two proteins and therefor for ICL repair. Additionally, we investigated mutations found in XPF in patients with FA, and found that these residues were important for the repair of ICLs, while they did not affect the function of XPF-ERCC1 in nucleotide excision repair. This is another important DNA repair pathway that deals with UV damage and bulky lesions and requires XPF-ERCC1. These separation of function mutations have shed light on the differential regulation of XPF-ERCC1 in multiple repair pathways. Finally, we have set up a mass spectrometry based screen to identify novel factors required for ICL repair. Taken together, this research has greatly enhanced our understanding of the molecular mechanism of the repair of ICLs. A more extensive knowledge of the ICL repair pathway and the function of the proteins involved will enhance our understanding of Fanconi anemia, and could also lead to the identification of potential targets that can be used sensitize cells to chemotherapeutic agents

Making the cut : How XPF-ERCC1 unhooks DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are highly toxic lesions that bind both strands of the DNA helix together, which prevents the DNA from unwinding. This blocks important cellular processes such as DNA replication and transcription. ICL inducing agents were among the first chemotherapeutic drugs, making use of the toxic nature of ICLs especially for fast dividing cells. Today, ICL-inducing agents are still the main line of treatment for many cancers. The repair mechanisms have long been elusive, but in recent years we have begun to understand the pathways that underlie ICL repair. Much of this knowledge has come from studying Fanconi anemia (FA), a rare genetic disorder that is characterized by an extreme cellular sensitivity to ICL-inducing agents. Repair of an ICL is initiated during DNA replication when a replication fork stalls at the damaged lesion. Subsequently, the FA pathway cooperates with a variety of DNA repair proteins, such as nucleases, translesion polymerases and recombinases to coordinate ICL repair. A critical step in the repair process are the dual incisions on one of the DNA strands, to release (or ‘unhook’) the ICL, but the exact mechanism of unhooking and the nucleases directly involved in this step have remained elusive. To investigate this we have made use of the Xenopus laevis egg extract. This unique system can recapitulate the replication-dependent repair of a DNA plasmid with a single site-specific ICL. Using this cell free system we have discovered that the structure specific endonuclease XPF-ERCC1 is absolutely required for the unhooking incisions to take place. Furthermore, the recruitment of XPF-ERCC1 to the site of damage is dependent on interaction with SLX4/FANCP. Recruitment of both XPF-ERCC1 and SLX4 is in turn promoted by the ubiquitination of FANCD2, a key step in the activation of the FA pathway. We furthermore shed insight into the interaction of SLX4 and XPF, by identifying residues on XPF that are crucial for the interaction of the two proteins and therefor for ICL repair. Additionally, we investigated mutations found in XPF in patients with FA, and found that these residues were important for the repair of ICLs, while they did not affect the function of XPF-ERCC1 in nucleotide excision repair. This is another important DNA repair pathway that deals with UV damage and bulky lesions and requires XPF-ERCC1. These separation of function mutations have shed light on the differential regulation of XPF-ERCC1 in multiple repair pathways. Finally, we have set up a mass spectrometry based screen to identify novel factors required for ICL repair. Taken together, this research has greatly enhanced our understanding of the molecular mechanism of the repair of ICLs. A more extensive knowledge of the ICL repair pathway and the function of the proteins involved will enhance our understanding of Fanconi anemia, and could also lead to the identification of potential targets that can be used sensitize cells to chemotherapeutic agents

Xenopus egg extract : A powerful tool to study genome maintenance mechanisms

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

Recruitment and positioning determine the specific role of the XPF-ERCC1 endonuclease in interstrand crosslink repair

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

XPF-ERCC1 acts in Unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4

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

A Salmonella virulence factor activates the NOD1/NOD2 signaling pathway.

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