The Escherichia coli recJ gene product was overproduced using a plasmid that carries the recJ gene downstream of a strong regulatable promoter and a strong ribosome-binding site. Overexpression of recJ produced a concomitant increase in the levels of single-stranded-DNAspecific nuclease activity present in crude cell extracts. This nuclease activity was purified to homogeneity and found to reside in a 60-kDa polypeptide. This polypeptide was induced with recJ overexpression and had the size and N-terminal amino acid sequence identical to the predicted RecJ protein sequence. The RecJ nuclease degraded linear single-stranded DNA but did not have exonuclease activity on linear doublestranded substrates or endonuclease activity on either singlestranded or double-stranded substrates. The RecJ exonuclease had greater activity on duplex DNA molecules with 5'-rather than 3'-single-stranded tails.

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli I-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed ,I-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3-to 12-fold, in good agreeement with previous mRNA analyses. When MATa cells were arrested in Gl with a-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL) promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.The regulation of DNA repair and recombination in the procaryote Escherichia coli has been extensively studied. One result of these studies has been the elucidation of the inducible SOS repair pathway, a component of which, the recA gene produpt, appears to control a number of genes involved in both the repair of DNA lesions and general DNA recombination (for a review, see reference 38). The regulation of such activities in eucaryotic systems has not yet been thoroughly elucidated. In Saccharomyces cerevisiae, mutations in three separate epistasis groups have been demonstrated to cause sensitivity to DNA-damaging agents such as UV light, X-rays, and chemical mutagens (12,14). However, each of these groups has a different phenotypic response to different kinds of damage, leading to the hypothesis that each group is involved in a separate repair pathway (13,25). Some mutations in one of these epistasis groups, the RAD50-57 group of genes, have been shown to be not only sensitive to the effects of ionizing radiation, but also to block meiotic recombination and to reduce some types of mitotic recombination (16,27). For this reason, the repair mediated by the RAD50-57 pathway has been postulated to be recombinational repair. Mutations in RAD54 and RAD52, the genes whose regulation we describe here, have been shown to be blocked in double-strand break repair (6,20,28). Additionally, rad52 mutations block significant levels of recombination in meiosis and yield inviable spores (15, 16). Although rad54 mutations previously had been reported to have little if any effect on meiosis, deletion mutations of the RAD54 gene have recently been isolated and shown to decrease bot...

Excision of deoxyribose-phosphate residues from enzymatically incised abasic sites in double-stranded DNA is required prior to gap-filling and ligation during DNA base excision-repair, and a candidate deoxyribophosphodiesterase (dRpase) activity has been identified in E. coli. This activity is shown here to be a function of the E. coli RecJ protein, previously described as a 5'-->3' single-strand specific DNA exonuclease involved in a recombination pathway and in mismatch repair. Highly purified preparations of dRpase contained 5'-->3' exonuclease activity for single-stranded DNA, and homogeneous RecJ protein purified from an overproducer strain had both 5'-->3' exonuclease and dRpase activity. Moreover, E. coli recJ strains were deficient in dRpase activity. The hydrolytic dRpase function of the RecJ protein requires Mg2+; in contrast, the activity of E. coli Fpg protein, that promotes the liberation of 5'-->3'Rp residues from DNA by beta-elimination, is suppressed by Mg2+. Several other E. coli nucleases, including exonucleases I, III, V, and VII, endonucleases I, III and IV and the 5'-->3' exonuclease function of DNA polymerase I, are unable to act as a dRpase. Nevertheless, E. coli fpg recJ double mutants retain capacity to repair abasic sites in DNA, indicating the presence of a back-up excision function.

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity ( (31,34,35,45).Recently, the gene encoding DNA polymerase II has been cloned (3,11,27), and its structural gene was identified as the SOS damage-inducible dinA gene (3,27). Pol II is part of the SOS regulon, having a LexA repressor operator site present in the promoter of the gene (3,27). Earlier, we had shown that Pol II could bypass a single site-directed abasic lesion in vitro, and levels of the enzyme in vivo exhibited a sevenfold increase

DNA exonucleases, enzymes that hydrolyze phosphodiester bonds in DNA from a free end, play important cellular roles in DNA repair, genetic recombination and mutation avoidance in all organisms. This article reviews the structure, biochemistry and biological functions of the 17 exonucleases currently identified in the bacterium Escherichia coli. These include the exonucleases associated with DNA polymerases I (polA), II (polB) and III (dnaQ/mutD), Exonucleases I (xonA/sbcB), III (xthA), IV, VII (xseAB), IX (xni/xgdG) and X (exoX), the RecBCD, RecJ, and RecE exonucleases, SbcCD endo/exonuclease, the DNA exonuclease activities of RNase T (rnt) and Endonuclease IV (nfo) and TatD. These enzymes are diverse in terms of substrate specificity and biochemical properties and have specialized biological roles. Most of these enzymes fall into structural families with characteristic sequence motifs, and members of many of these families can be found in all domains of life.