RecJ exonuclease: substrates, products and interaction with SSB

Abstract

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5′ single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg2+ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg2+ confirmed that substrates with 5′ tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading ∼1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5′ phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.

Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli

“…Despite a commonly held expectation that RecQ helicase would be needed to unwind the dsDNA into ssDNA that is the substrate for exonucleolytic processing by RecJ, which is an ssDNA-specific exonuclease, we discovered that RecQ helicase was stimulatory but not essential. Instead, resection is via a capacity of RecJ to degrade one strand of dsDNA (Han et al 2006). These findings are, in fact, consistent with genetic analyses that established that mutation of recJ results in a greater reduction of recombination by the RecF pathway than mutation of recQ (Luisi-DeLuca et al 1989;Sawitzke and Stahl 1992); this in vivo observation could be a consequence of redundancy in helicase function provided by helicases II and IV (Mendonca et al 1995).…”

“…RecJ exonuclease prefers ssDNA substrates with 59-tails that are longer than 7 nucleotides (nt); however, it does display a limited slower degradation of dsDNA that previously had been shown to extend ;10 nt into the duplex region of a tailed DNA substrate (Han et al 2006). This finding indicates that RecJ has an intrinsic capacity to resect duplex DNA to produce 39-tailed ssDNA that is the ideal substrate for DNA strand invasion.…”

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

“…2, lanes 11 and 12) in the presence of MgCl 2 . Because RecJ is known to be a 5Ј-3Ј exonuclease (14,15,18,19), these results indicated that TTHA0118 had 5Ј-3Ј exonuclease activity for ssDNA.…”

“…One important point is the polarity of exonuclease degradation by NrnA, which was reported to be 3Ј to 5Ј (21). However, RecJ exonuclease has 5Ј to 3Ј polarity (14,15,18,19). In addition, the detailed phenotype of the RecJ-like gene disruptant has not been reported until now.…”

Genetic and biochemical characterization ofDrosophilaSnipper: A promiscuous member of the metazoan 3′hExo/ERI-1 family of 3′ to 5′ exonucleases

“…Our studies show that Snp is a promiscuous 39-59 exonuclease with broad specificity toward both RNA and DNA substrates, although it exhibits highest affinity toward double-stranded substrates with a 39 flank. Similar to many processive 39-59 exonucleases such as the DNases Exo I (Breyer and Matthews 2000), RecJ (Han et al 2006), and Exo X (Viswanathan and Lovett 1999), and the RNases PARN (Wu et al 2005), PNPase (Symmons et al 2002), RNase R (Cheng and Deutscher 2005), Snp is processive on hairpin DNAs and RNAs with at least three to five unpaired nucleotides in the 39 flank and nonprocessive on singlestranded DNAs and RNAs. Snp is highly active toward histone stem-loop RNA in vitro and unlike 39hExo (which has an N-terminal SAP domain), Snp readily digests through the stem portion of the histone RNA hairpin.…”