RecN Is a Cohesin-like Protein That Stimulates Intermolecular DNA Interactions in Vitro

Abstract

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...

DNA compaction in the early part of the SOS response is dependent on RecN and RecA

“…In vitro, purified RecN protein has been found to have a weak ATPase activity and DNA-bridging activity (Grove et al, 2009;Reyes et al, 2010). We found that cells lacking RecN protein did not compact or decompact the nucleoid after irradiation with a UV dose of 3 J m 22 (Fig.…”

Oxidative Stress Resistance inDeinococcus radiodurans

“…All four proteins, in particular RecJ, are required for normal cellular growth and recombinational repair (49). Recombinational repair is facilitated by RecN (DR1477), an SMC family member which tethers DNA molecules in a cohesin-like fashion and prevents the separation of DNA fragments (518). In B. subtilis, RecN is one of the first proteins recruited to DSBs (531).…”

“…In B. subtilis, RecN is one of the first proteins recruited to DSBs (531). In vitro, D. radiodurans RecN also stimulates the intermolecular ligation of linear DNA molecules in the presence of DNA ligase (518).…”

RecA Protein Recruits Structural Maintenance of Chromosomes (SMC)-like RecN Protein to DNA Double-strand Breaks

“…These results may reflect species-specific attributes of E. coli and B. subtilis HR pathways. The purified B. subtilis RecN (and also D. radiodurans RecN) binds to DNA and has DNA-stimulated ATPase activity in vitro (54,55). Unfortunately, it has been difficult to purify E. coli RecN because it is relatively insoluble and highly susceptible to degradation (data not shown).…”