Generation of DNA double-strand breaks by two independent enzymatic activities in nuclear extracts.
We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.
Blanco
Miguel G
MG
Departamento de Bioquímica e Bioloxía Molecular, Facultade de Bioloxía, Universidade de Santiago de Compostela, Galicia 15782, Spain.
Boán
Francisco
F
Barros
Paula
P
Castaño
José G
JG
Gómez-Márquez
Jaime
J
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002474:Cell-Free System; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D004533:Egtazic Acid / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D009841:Oligonucleotides; D004533:Egtazic Acid; D004247:DNA; D008274:Magnesium