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

Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6023324

Fraser, M J; Huang, X; Hatahet, Z

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.

• Research Organization: Univ. of Montreal, Quebec
• OSTI ID: 6023324
• Report Number(s): CONF-870644-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 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

Related Subjects

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