SummaryThe 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.

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 ( 405 RRR 407 ) within the GABA A R ␤3-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 ␤3-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 405 RRR 407 motif. Thus, AP2 binding to an RRR motif in the GABA A R ␤3-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.

Escherichia coli

The bacterial recombinase, RecA protein, plays a central role in maintenance of genome stability. It generally functions as a RecA nucleoprotein filament formed on DNA (1). RecA has three identified functions in the cell. First, it participates directly in all recombination processes. Filaments of Escherichia coli RecA protein form most readily on ssDNA; these filaments can then catalyze DNA pairing and strand exchange with a homologous duplex DNA (2). A RecA filament complexed with an oligonucleotide can invade duplex DNA and pair the oligonucleotide with a complementary sequence within a much longer duplex, forming a displacement loop (D-loop) (2). Second, RecA filaments play a central role in the induction of the SOS response (3). In brief, RecA filaments assembled on ssDNA generated by stalled replication forks bind and stimulate the autocatalytic cleavage of the LexA repressor. This co-protease function for RecA inactivates LexA, which leads to the induction of SOS. Third, RecA filaments play two roles in activating the translesion DNA synthesis function of DNA polymerase V. If cellular genomic replication is not restored after 30 -60 min of SOS response, polymerase V opens a final, mutagenic phase of SOS. The UmuD subunit of polymerase V is autocatalytically cleaved, again facilitated by interaction with RecA filaments, to create UmuDЈ. The weakly active UmuDЈ 2 ⅐UmuC complex undergoes a final activation step with the transfer of a RecA subunit from the 3Ј-proximal end of a RecA filament to form the activated polymerase V enzyme consisting of UmuDЈ 2 ⅐UmuC/RecA (4, 5). The presence of RecA as a subunit of active polymerase V is the only known activity where RecA exhibits a function when it is not part of a filament.Many other proteins interact with RecA protein filaments on DNA, and many of these serve to regulate almost every aspect of RecA function (6). The regulators include DinI (7), RecX (8 -10), RdgC (11), PsiB (12), RecFOR (13-15), UvrD (16), and RecBCD (17-19), a list that will doubtlessly grow. In all, more than a dozen known proteins interact with RecA and help coordinate its functions with many aspects of DNA metabolism. A number of RecA partner proteins bind within the helical groove of active RecA nucleoprotein filaments. These include LexA (20,21), UmuD (22), RecX (20), and DinI.2 The LexA and UmuD proteins (along with the bacteriophage repressor (23)) undergo autocatalytic cleavage in this environment. The regulation of RecA is not limited to bacteria-encoded functions. The PsiB protein is a product of conjugative F-plasmids, expressed early in conjugation to suppress the SOS response in the recipient cell (12,24,25). Additional proteins that interact with RecA are encoded by bacteriophages, including the bacteriophage P1.Almost from the moment it was described in 1951 (26), bacteriophage P1 has been a workhorse of molecular biology. It is now used largely for generalized transduction applied to strain construction (27,28). Its genome (93.6 kbp; ϳ117 genes (27)) is packaged in phage particl...