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Title: How Neurospora endo-exonuclease might act in recombinational double strand break DNA repair

Abstract

Neurospora endo-exonuclease (EE), an enzyme with both single strand (SS) specific endonuclease activity and processive exonuclease activity with double strand (ds) DNA, has been further characterized. EE made random nicks in supercoiled (ccc) pBR322 DNA and converted the relaxed DNA to the linear form. Electron microscopic examination of supercoiled PM2 and linear T7 ds-DNAs treated with EE revealed ds-breaks with termini that remained juxtaposed. When pBR322 DNA was linearized with unique site restriction nucleases and treated with EE, ladders of DNA fragments were detected in agarose gels indicating site-specific ds-breaks. To examine this further, Sca I linearized pBR322 was labelled at either the 5'-termini with ..gamma..-/sup 32/P-ATP and polynucleotide kinase or the 3'-termini with ..cap alpha..-/sup 32/P-dTTP and E. coli DNA polymerase I. EE cut 100 times faster at the 5'- than at the 3'-termini indicating that its exonuclease activity had a strong preference for acting 5' to 3'. The sequences of nucleotides at some preferred break sites were determined. In 3 cases examined, high homology was observed for 6 nucleotides, especially on the 5'-side of the breaks. The results indicate that EE might function in recombinational ds-break repair by assembling in dimeric or multimeric form at damage-induced ds-breaks ormore » create site-specific ds-breaks at which its exonuclease acts processively on the termini to degrade the 5'-p-terminated strands while generating 3'-OH-terminated ss-tails for switching into homologous duplexes.« less

Authors:
; ;
Publication Date:
Research Org.:
Univ. of Montreal, Quebec
OSTI Identifier:
6023324
Report Number(s):
CONF-870644-
Journal ID: CODEN: FEPRA
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DNA REPAIR; BIOCHEMISTRY; ENDONUCLEASES; BIOLOGICAL FUNCTIONS; ENZYME ACTIVITY; ATP; DNA; DNA POLYMERASES; DNA SEQUENCING; GENE RECOMBINATION; NEUROSPORA; PHOSPHORUS 32; PHOSPHOTRANSFERASES; STRAND BREAKS; TRACER TECHNIQUES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOLOGICAL RECOVERY; BIOLOGICAL REPAIR; CHEMISTRY; DAYS LIVING RADIOISOTOPES; DNA-ASE; ENZYMES; ESTERASES; FUNCTIONS; FUNGI; HYDROLASES; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; NUCLEI; NUCLEIC ACIDS; NUCLEOTIDES; NUCLEOTIDYLTRANSFERASES; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; PHOSPHODIESTERASES; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; PLANTS; POLYMERASES; RADIOISOTOPES; RECOVERY; REPAIR; STRUCTURAL CHEMICAL ANALYSIS; TRANSFERASES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Fraser, M J, Huang, X, and Hatahet, Z. How Neurospora endo-exonuclease might act in recombinational double strand break DNA repair. United States: N. p., 1987. Web.
Fraser, M J, Huang, X, & Hatahet, Z. How Neurospora endo-exonuclease might act in recombinational double strand break DNA repair. United States.
Fraser, M J, Huang, X, and Hatahet, Z. Fri . "How Neurospora endo-exonuclease might act in recombinational double strand break DNA repair". United States.
@article{osti_6023324,
title = {How Neurospora endo-exonuclease might act in recombinational double strand break DNA repair},
author = {Fraser, M J and Huang, X and Hatahet, Z},
abstractNote = {Neurospora endo-exonuclease (EE), an enzyme with both single strand (SS) specific endonuclease activity and processive exonuclease activity with double strand (ds) DNA, has been further characterized. EE made random nicks in supercoiled (ccc) pBR322 DNA and converted the relaxed DNA to the linear form. Electron microscopic examination of supercoiled PM2 and linear T7 ds-DNAs treated with EE revealed ds-breaks with termini that remained juxtaposed. When pBR322 DNA was linearized with unique site restriction nucleases and treated with EE, ladders of DNA fragments were detected in agarose gels indicating site-specific ds-breaks. To examine this further, Sca I linearized pBR322 was labelled at either the 5'-termini with ..gamma..-/sup 32/P-ATP and polynucleotide kinase or the 3'-termini with ..cap alpha..-/sup 32/P-dTTP and E. coli DNA polymerase I. EE cut 100 times faster at the 5'- than at the 3'-termini indicating that its exonuclease activity had a strong preference for acting 5' to 3'. The sequences of nucleotides at some preferred break sites were determined. In 3 cases examined, high homology was observed for 6 nucleotides, especially on the 5'-side of the breaks. The results indicate that EE might function in recombinational ds-break repair by assembling in dimeric or multimeric form at damage-induced ds-breaks or create site-specific ds-breaks at which its exonuclease acts processively on the termini to degrade the 5'-p-terminated strands while generating 3'-OH-terminated ss-tails for switching into homologous duplexes.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 46:6,
place = {United States},
year = {1987},
month = {5}
}

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