Stabilization of GABA(A) receptors at endocytic zones is mediated by an AP2 binding motif within the GABA(A) receptor ß3 subunit.

The strength of synaptic inhibition can be controlled by the stability and endocytosis of surface and synaptic GABA(A) receptors (GABA(A)Rs), but the surface receptor dynamics that underpin GABA(A)R recruitment to dendritic endocytic zones (EZs) have not been investigated. Stabilization of GABA(A)Rs at EZs is likely to be regulated by receptor interactions with the clathrin-adaptor AP2, but the molecular determinants of these associations remain poorly understood. Moreover, although surface GABA(A)R downmodulation plays a key role in pathological disinhibition in conditions such as ischemia and epilepsy, whether this occurs in an AP2-dependent manner also remains unclear. Here we report the characterization of a novel motif containing three arginine residues (405RRR407) within the GABA(A)R ß3-subunit intracellular domain (ICD), responsible for the interaction with AP2 and GABA(A)R internalization. When this motif is disrupted, binding to AP2 is abolished in vitro and in rat brain. Using single-particle tracking, we reveal that surface ß3-subunit-containing GABA(A)Rs exhibit highly confined behavior at EZs, which is dependent on AP2 interactions via this motif. Reduced stabilization of mutant GABA(A)Rs at EZs correlates with their reduced endocytosis and increased steady-state levels at synapses. By imaging wild-type or mutant super-ecliptic pHluorin-tagged GABA(A)Rs in neurons, we also show that, under conditions of oxygen-glucose deprivation to mimic cerebral ischemia, GABA(A)Rs are depleted from synapses in dendrites, depending on the 405RRR407 motif. Thus, AP2 binding to an RRR motif in the GABA(A)R ß3-subunit ICD regulates GABA(A)R residency time at EZs, steady-state synaptic receptor levels, and pathological loss of GABA(A)Rs from synapses during simulated ischemia.

Creating directed double-strand breaks with the Ref protein: a novel RecA-dependent nuclease from bacteriophage P1.

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 Å resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded ß-hairpin that is sandwiched between several α-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

Less is more: Neisseria gonorrhoeae RecX protein stimulates recombination by inhibiting RecA.

Escherichia coli RecX (RecX(Ec)) is a negative regulator of RecA activities both in the bacterial cell and in vitro. In contrast, the Neisseria gonorrhoeae RecX protein (RecX(Ng)) enhances all RecA-related processes in N. gonorrhoeae. Surprisingly, the RecX(Ng) protein is not a RecA protein activator in vitro. Instead, RecX(Ng) is a much more potent inhibitor of all RecA(Ng) and RecA(Ec) activities than is the E. coli RecX ortholog. A series of RecX(Ng) mutant proteins representing a gradient of functional deficiencies provide a direct correlation between RecA(Ng) inhibition in vitro and the enhancement of RecA(Ng) function in N. gonorrhoeae. Unlike RecX(Ec), RecX(Ng) does not simply cap the growing ends of RecA filaments, but it directly facilitates a more rapid RecA filament disassembly. Thus, in N. gonorrhoeae, recombinational processes are facilitated by RecX(Ng) protein-mediated limitations on RecA(Ng) filament presence and/or length to achieve maximal function.

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis.

The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo. We have combined the K72R variant of RecA with another mutation, RecA E38K (recA730). In vitro, the double mutant RecA E38K/K72R (recA730,2201) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro.

Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

SSB antagonizes RecX-RecA interaction.

The RecX protein of Escherichia coli inhibits the extension of RecA protein filaments on DNA, presumably by binding to and blocking the growing filament end. The direct binding of RecX protein to single-stranded DNA is weak, and previous reports suggested that direct binding to DNA did not explain the effects of RecX. We now demonstrate that elevated concentrations of SSB greatly moderate the effects of RecX protein. High concentrations of the yeast RPA protein have the same effect, suggesting that the effect is not species-specific or even specific to bacterial SSB proteins. A direct SSB-RecX interaction is thus unlikely. We suggest that SSB is blocking access to single-stranded DNA. The evident competition between RecX and SSB implies that the mechanism of RecX action may involve RecX binding to both RecA protein and to DNA. We speculate that the interaction of RecX protein and RecA may enable an enhanced DNA binding by RecX protein. The effects of SSB are increased if the SSB C terminus is removed.