The bacterial RecN protein is involved in the recombinational repair of DNA double-stranded breaks, and recN mutants are sensitive to DNA-damaging agents. Little is known about the biochemical function of RecN. Protein sequence analysis suggests that RecN is related to the SMC (structural maintenance of chromosomes) family of proteins, predicting globular N-and C-terminal domains connected by an extensive coil-coiled domain. The N-and C-domains contain the nucleotide-binding sequences Walker A and Walker B, respectively. We have purified the RecN protein from Deinococcus radiodurans and characterized its DNA-dependent and DNA-independent ATPase activity. The cellular genome maintenance processes of DNA replication, recombination, and repair are highly interconnected, sharing multiple pathways and common enzymes. A physical understanding of the function of key enzymes involved in genome maintenance processes is critical for elucidating the basic DNA metabolic strategies of cells. The primary function of homologous DNA recombination in mitotic cells as well as bacterial cells is the nonmutagenic repair of replication forks that have stalled or collapsed at noncoding lesions or breaks (1-3). When a replication fork encounters a break in the phosphodiester backbone of one template strand, a double-stranded break (DSB) 2 results (4). Recombinational DSB repair pathways promote strand invasion to regenerate a fork structure. In Escherichia coli, these pathways include the helicase and nuclease functions of the RecBCD complex, and the RecA protein. RecN, among various other proteins, is required for DSB repair in bacteria. Existing DSB repair models do not encompass RecN, because no clear function has been ascribed to the protein as yet.The recN gene of E. coli (also known as radB (5)) was identified by two groups over 20 years ago. Lloyd et al. (6) isolated a mutation (rec-259) that caused increased sensitivity to UV light in a recBC sbcB background. Independently, Sargentini and Smith (7) isolated the radB101 mutation, which caused sensitivity to ␥-irradiation and mitomycin C. Expression of the recN gene is regulated by the LexA repressor (8, 9), and the RecN protein is one of the most abundantly produced proteins induced as part of the SOS response to DNA damage (10). Once produced in response to damage, RecN protein has a very short half-life (ϳ10 min), because it apparently is rapidly proteolyzed by the ClpXP protease system (11).The recN gene product was originally placed in the RecF-dependent gap repair pathway, but substantial evidence suggests that RecN is also involved in the RecBCD DSB repair pathway. Unlike other members of the RecF pathway, recN mutants are quite sensitive to ionizing radiation or mitomycin C, and the repair of multiple, site-specific DSBs does not occur in the absence of RecN (8,12,13). The recN gene is also required for the suppression of chromosomal rearrangements and deletions (12) during the repair of a single DSB. Given this wealth of genetic data, the RecN protein is clearly involve...

RecN is a cohesin-like protein involved in DNA double-strand break repair in bacteria. The RecA recombinase functions to mediate repair via homologous DNA strand invasion to form D-loops. Here we provide evidence that the RecN protein stimulates the DNA strand invasion step of RecA-mediated recombinational DNA repair. The intermolecular DNA tethering activity of RecN protein described previously cannot fully explain this novel activity since stimulation of RecA function is species-specific and requires RecN ATP hydrolysis. Further, DNA-bound RecA protein increases the rate of ATP hydrolysis catalysed by RecN during the DNA pairing reaction. DNA-dependent RecN ATPase kinetics are affected by RecA protein in a manner suggesting a specific order of protein–DNA assembly, with RecN acting after RecA binds DNA. We present a model for RecN function that includes presynaptic stimulation of the bacterial repair pathway perhaps by contributing to the RecA homology search before ternary complex formation.

Escherichia coli dinD is an SOS gene up-regulated in response to DNA damage. We find that the purified DinD protein is a novel inhibitor of RecA-mediated DNA strand exchange activities. Most modulators of RecA protein activity act by controlling the amount of RecA protein bound to single-stranded DNA by affecting either the loading of RecA protein onto DNA or the disassembly of RecA nucleoprotein filaments bound to singlestranded DNA. The DinD protein, however, acts postsynaptically to inhibit RecA during an on-going DNA strand exchange, likely through the disassembly of RecA filaments. DinD protein does not affect RecA single-stranded DNA filaments but efficiently disassembles RecA when bound to two or more DNA strands, effectively halting RecA-mediated branch migration. By utilizing a nonspecific duplex DNA-binding protein, YebG, we show that the DinD effect is not simply due to duplex DNA sequestration. We present a model suggesting that the negative effects of DinD protein are targeted to a specific conformational state of the RecA protein and discuss the potential role of DinD protein in the regulation of recombinational DNA repair.The Escherichia coli SOS response is a coordinately regulated network of genes induced in reaction to heavy or persistent DNA damage (1). The SOS regulon is repressed by the LexA protein, which is inactivated as a repressor upon cellular stress such as heavy DNA damage. The cellular signal for SOS induction is the RecA protein bound to single-stranded DNA (ssDNA). 2RecA is the central DNA recombinase in bacteria and carries out several distinct functions when activated (2). The most appreciated function of RecA is the catalysis of homologous DNA recombination crucial to the generation of genetic diversity. However, based on frequency of use, the primary role of RecA lies in the multiple pathways for the recombinational repair of stalled DNA replication forks. Through much in vitro work, it has become increasingly apparent that the RecA protein is under considerable control by a network of proteins that function to modulate when and where RecA protein binds to DNA (3). The PsiB protein has recently been shown to bind to free RecA protein, effectively inhibiting RecA from nucleating onto ssDNA (4). RecA is also inhibited from nucleating onto ssDNA bound by the single-stranded DNA-binding protein (SSB) (5). The SSB-imposed inhibition is relieved by the action of the RecF, RecO, and RecR proteins (3). Following nucleation, RecA protein protomers assemble into a nucleoprotein filament, extending cooperatively in the 5Ј to 3Ј direction. The RdgC protein can inhibit RecA binding to ssDNA and can interfere with homologous DNA pairing by binding to duplex DNA (6, 7). Some proteins shown to dismantle RecA filaments are known DNA translocases, such as UvrD (8) and PcrA helicases (9). Filament extension can be blocked through the action of the RecX protein (10), whereas the DinI protein antagonizes the function of RecX by stabilizing RecA filaments, inhibiting filament end-dependent disassem...

A series of cystargolide-based β-lactone analogues containing nitrogen atoms at the Pz portion of the scaffold were prepared and evaluated as proteasome inhibitors, and for their cytotoxicity profile toward several cancer cell lines. Inclusion of one, two or even three nitrogen atoms at the Pz portion of the cystargolide scaffold is well tolerated, producing analogues with low nanomolar proteasome inhibition activity, in many cases superior to carfilzomib. Additionally, analogue 8g , containing an ester and pyrazine group at Pz, was shown to possess significant activity toward RPMI 8226 cells (IC 50 = 21 nM) and to be less cytotoxic toward the normal tissue model MCF10A cells than carfilzomib.

Deinococcus radioduransis a radiation resistant organism with the ability to survive hundreds of DNA double‐strand breaks. The compacity to withstand such a detrimental type of DNA break stems from the efficiency of the DNA repair machinery of this organism. D. radioduransrecombination mediator proteins RecO and RecR have been shown to be required for radiation resistance in this organism. In E. colithe RecOR proteins have been reported to stimulate RecA filament nucleation onto SSB‐bound single‐stranded DNA (ssDNA) through displacement of the SSB protein. However, how D. radioduransRecO and RecR proteins work to mediate RecA filament formation is not well understand. In this study, we further elucidate the biochemical mechanism of RecOR function; the kinetic evidence demonstrates that D. radiodurans RecOR alter the rate of ATP hydrolysis by the RecA protein when bound to ssDNA regardless of the presence of SSB protein. This effect has not been previously described for other homologous recombination systems. We believe that D. radioduransRecOR functions to stimulate RecA nucleation to ssDNA directly and independently of SSB protein. Potential mechanisms for this kinetic stimulation will be discussed.