The actions of Neurospora endo-exonuclease on double strand DNAs.
Neurospora crassa endo-exonuclease, an enzyme implicated in recombinational DNA repair, was found previously to have a distributive endonuclease activity with a high specificity for single strand DNA and a highly processive exonuclease activity. The activities of endo-exonuclease on double strand DNA substrates have been further explored. Endo-exonuclease was shown to have a low bona fide endonuclease activity with completely relaxed covalently closed circular DNA and made site-specific breaks in linear double strand DNA at a low frequency while simultaneously generating a relatively high level of single strand breaks (nicks) in the DNA. Sequencing at nicks induced by endo-exonuclease in pBR322 restriction fragments showed that the highest frequency of nicking occurred at the mid-points of two sites with the common sequence, p-AGCACT-OH. In addition, sequencing revealed less frequent nicking at identical or homologous hexanucleotide sequences in all other 54 cases examined where these sequences either straddled the break site itself or were within a few nucleotides on either side of the break site. The exonucleolytic action of endo-exonuclease on linear DNA showed about 100-fold preference for acting in the 5' to 3' direction. Removal of the 5'-terminal phosphates substantially reduced this activity, internal nicking, and the ability of endo-exonuclease to generate site-specific double strand breaks. On the other hand, nicking of the dephosphorylated double strand DNA with pancreatic DNase I stimulated the exonuclease activity by almost 5-fold, but no stimulation was observed when the DNA was nicked by Micrococcal nuclease. Thus, 5'-p termini either at double strand ends or at nicks in double strand DNA are entry points to the duplex from which endo-exonuclease diffuses linearly or "tracks" in the 5' to 3' direction to initiate its major endo- and exonucleolytic actions. The results are interpreted to show how it is possible for endo-exonuclease to generate single strand DNA for switching into a homologous duplex either at a nick or while remaining bound at a double strand break in the DNA. Such mechanisms are consistent with current models for recombinational double strand break repair in eukaryotes.
Fraser
M J
MJ
Department of Biochemistry, McGill University, Montreal, Canada.
Hatahet
Z
Z
Huang
X T
XT
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004270:DNA, Circular / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013379:Substrate Specificity
D004270:DNA, Circular; D004271:DNA, Fungal; D004247:DNA; D004720:Endonucleases; D005092:Exonucleases; C020104:endoexonuclease