Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli

Abstract

Immunochemical cross-reaction between the endo-exonuclease of Neurospora crassa, an enzyme previously implicated in recombination and recombinational DNA repair, and the recC-encoded polypeptide of Esherichia coli has been detected by immunoblotting extracts of strains of E. coli having a deletion that includes the recBCD genes but carrying multicopy plasmids bearing all three of the recBCD genes or only one or two of these genes. It was predicted that homology would also be found at the amino acid sequence level between the recC polypeptide and both nuclear and mitochondrial endo-exonucleases of Saccharomyces cerevisiae, which cross-react with antibodies raised to the N. crassa endo-exonuclease. Since the gene for the S. cerevisiae mitochondrial enzyme, NUCI, has been cloned and sequenced and the predicted amino acid sequence is known, this sequence was aligned with the predicted amino acid sequence of the recC polypeptide. Extensive homology was found by aligning 306 of the 329 amino acids of the yeast mitochondrial nuclease sequence with the carboxy-terminal one-quarter of the amino acid sequence of the recC polypeptide.Possible roles for endo-exonuclease of Neurospora crassa in recombination and recombinational repair of nuclear and mitochondrial DNAs have been proposed previously, on the basis of strong similarities in the enzymic activities of this enzyme and those of the recBCD-encoded nuclease (exonuclease V) of Escherichia coli (4) and deficiencies in active endo-exonuclease in nuclei and mitochondria of the mutagen-sensitive uvs-3 mutant of Neurospora sp., which may be abnormal in recombination (12,15). In addition, an immunochemically related nuclear endo-exonuclease of 72 kilodaltons (kDa) was shown to be very deficient in the recombination and recombinational double-strand DNA break repair-deficient rad52 mutant of Saccharomyces cerevisiae (5, 6). The nuclear endo-exonuclease is encoded by an essential gene which has been cloned and is currently being sequenced (T. Y.-K. Chow, unpublished observations). A second endo-exonuclease, of 38 kDa, which is also immunochemically related to the N. crassa endo-exonuclease is present in the S. cerevisiae mitochondria (7). It is encoded by the nonessential NUCI gene, which has recently been cloned and sequenced (19).In contrast to E. coli exonuclease V, the N. crassa endo-exonuclease is sugar nonspecific, acting on both DNA and RNA, and although it is not ATP dependent or ATP stimulated in its actions, it does possess a distributive single-stranded DNA-specific endonuclease activity and a highly processive exonuclease activity with linear doublestranded DNA, like exonuclease V (4). The latter activity in vitro can generate long single-stranded tails and long gaps in duplex DNA, which would be potentially recombinogenic substrates if also generated in vivo. Under slightly different conditions in vitro, N. crassa endo-exonuclease has been shown recently to make site-specific single-and doublestrand breaks in linear, but not in covalently closed circular, V (17)....

Biochemistry of homologous recombination in Escherichia coli.

“…An independently isolated class of recA mutations displaying constitutive coprotease activity were found to map in the two regions that also include the recA441 and recA803 mutations (amino acid residues 25 to 39 and 298 to 301) (635). 184), between the loops proposed to be involved in DNA binding (607) (Fig. 7).…”

Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae

“…Because of the dependence of the RAD52 gene product (Chow & Resnick, 1988), the elevated levels of immunoprecipitable nuclease in meiosis (Resnick et al+, 1984), and the observed cross-reactivity of the N. crassa antibodies to the E. coli recC gene product (Fraser et al+, 1990), the yNucR endo-exonuclease was proposed to be important in DNA recombination and/or DNA repair (Chow & Kunz, 1991;Moore et al+, 1993;Sadekova & Chow, 1996;Asefa et al+, 1998)+ RAD52 and other members of the RAD52 epistasis group are required for X-ray damage repair and various types of intra-and interchromosomal mitotic recombination re-pair+ RAD52 is also required for repair of doublestranded breaks during induction of HO-endonuclease and is essential during sporulation (Friedberg et al+, 1995)+…”

Meiosis in Mycelial Fungi

“…b Products of the first meiotic division fail to separate completely, resulting in spindles with four poles at the second meiotic division. c Although uvs3 strains are deficient in an endo-exonuclease with similarities to recC of Escherichia coli (Fraser et al 1990), uvs3 does not appear to be a meiotic mutant, since development is arrested prior to karyogamy (Schroeder 1970 pers. comm.).…”