The actions of Neurospora endo-exonuclease on double strand DNAs.

M. J. Fraser, Zafer Hatahet, X. T. Huang

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)13093-13101
Number of pages9
JournalThe Journal of biological chemistry
Volume264
Issue number22
StatePublished - Jan 1 1989

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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