Structure of RecJ Exonuclease Defines Its Specificity for Single-stranded DNA

Abstract

RecJ is a single-stranded DNA (ssDNA)-specific 5-3 exonuclease that plays an important role in DNA repair and recombination. To elucidate how RecJ achieves its high specificity for ssDNA, we determined the entire structures of RecJ both in a ligand-free form and in a complex with Mn 2؉ or Mg 2؉ by x-ray crystallography. The entire RecJ consists of four domains that form a molecule with an O-like structure. One of two newly identified domains had structural similarities to an oligonucleotide/oligosaccharide-binding (OB) fold. The OB fold domain alone could bind to DNA, indicating that this domain is a novel member of the OB fold superfamily. The truncated RecJ containing only the core domain exhibited much lower affinity for the ssDNA substrate compared with intact RecJ. These results support the hypothesis that these structural features allow specific binding of RecJ to ssDNA. In addition, the structure of the RecJ-Mn 2؉ complex suggests that the hydrolysis reaction catalyzed by RecJ proceeds through a two-metal ion mechanism.All of the organisms possess numerous pathways to repair DNA damages to maintain the integrity of the information contained in the genome. The single-stranded DNA (ssDNA) 2 processing exonuclease RecJ has been implicated in many of these repair pathways. RecJ was originally identified as an essential gene for the RecF pathway of homologous recombination (1). RecJ produces 3Ј-ssDNA tails, which are required to initiate recombination from a double-stranded break (2). RecJ is also an exonuclease that mediates the excision step during mismatch repair (3, 4) and degrades abasic residues generated during base excision repair (5). Almost all eubacteria and archaea contain RecJ orthologs (6). These observations suggest that RecJ plays an important role in DNA repair.RecJ is the only known ssDNA-specific 5Ј exonuclease. It specifically hydrolyzes ssDNA in the 5Ј to 3Ј direction, which is dependent on metal cations (7). It processively degrades its substrate at ϳ700 (8) or 1,000 (9) nucleotides/single binding event and appears to have a site that can bind seven nucleotides (9).The crystal structure of a truncated Thermus thermophilus RecJ (ttRecJ) shows that the core domain of ttRecJ (cd-ttRecJ) (residues 40 -463) consists of two domains that are interconnected by a long helix, forming a central cleft (10). Mn 2ϩ in the active site is located on the wall of the cleft and is coordinated by conserved residues characteristic of a family of phosphoesterases that includes RecJ proteins. Although cd-ttRecJ contains the conserved residues of DHH (motifs I-IV) and DHHA1 motifs (11) (supplemental Fig. S1), the entire structure including the N-and C-terminal regions has not been determined.To elucidate how RecJ achieves its high specificity for ssDNA, we determined the intact ttRecJ structures (666 residues) both in a ligand-free form and in a complex with Mn 2ϩ or Mg 2ϩ at 2.15-2.50 Å resolution. Intact ttRecJ consists of four domains that form a molecule with an O-like structure. Interestingly, one of two ne...

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

“…In addition, we targeted B. subtilis NrnA to reexamine the polarity of its exonuclease activity (21). Furthermore, we used ttRecJ (38) and cd-ttRecJ (37) from T. thermophilus HB8 as a control to highlight differences between RecJ and RecJ-like proteins.…”

“…This can be explained by the fact that cd-ttRecJ lacks the domain containing a novel OB-fold (39), that is in the C-terminal region of ttRecJ (Figs. 1B and 6C) (38). Lack of the OB-fold-containing domain might also explain the low substrate affinity of TTHA0118, but not the high affinity of Mpn140.…”

“…T. thermophilus HB8 contains three DHH superfamily proteins: RecJ (TTHA1167, ttRecJ), a putative poly(A) polymerase (TTHA0831), and a RecJ-like protein (TTHA0118). Our group determined the crystal structures of the core domain (residues 40 -463) of ttRecJ, designated cd-ttRecJ (37), and the intact ttRecJ (38). The intact ttRecJ structure has four domains which form a molecule with an O-like structure.…”

RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination

“…The crystal structure of full length Thermus thermophilus RecJ revealed a hole that accommodates a ssDNA strand, but not dsDNA, located on one side of an oligonucleotide-binding (OB) domain, offering a structural explanation and mechanism for ssDNA strand specificity of RecJ exonuclease (57). Such a mechanism can explain why RecJ-mediated resection of dsDNA frequently stopped upstream of GC-rich regions (Fig.…”

O Antigen Is the Receptor of Vibrio cholerae Serogroup O1 El Tor Typing Phage VP4

“…In fact, in addition to the O-antigen receptor genes, we found that recJ may also be related to VP4 infection, because complementing the transposon mutant of N16961 with intact recJ restored sensitivity to VP4 infection (data not shown). The recJ gene is recognized as being involved in base excision repair, mismatch repair, homologous recombination (58), and the rescue of arrested replication forks (59), and it is also the only 5=-3= exonuclease specific for single-stranded DNA (ssDNA) in most prokaryotic species (60,61). In Acinetobacter baylyi, the recJ DNase strongly suppresses short foreign DNA fragments from integrating into genomic DNA (62).…”