Mouse testicular extracts process DNA double-strand breaks efficiently by DNA end-to-end joining.
DNA double-strand break (DSB) processing was studied in mouse testicular extracts using a defined DSB created by cleaving supercoiled pUC12 DNA at a unique site as the substrate, and analysing the processed DNA by gel electrophoresis. Our results demonstrated that enzymatic activity in the extracts promoted multimerization of DNA and suppressed its circularization. This was distinctly different from T4 DNA ligase activity in the control and therefore the process must be more complex than simple ligation. Efficiency of this end-to-end joining was ATP and Mg(2+)-dependent and was much higher with cohesive (especially with 5') than with blunt ends. On recleaving, the joining was predominantly faithful, especially for cohesive ends; but a detectable fraction of DNA had undergone end-processed joining causing junctional deletions, mostly with blunt ends. Redigestion of end-joined products from time course experiments established that the end-deleted joining occurred concurrent to the faithful joining. Junctional segments were cloned and their restriction analysis confirmed the presence of large deletions from both the sides. These results suggested the association of an end-processing activity (exonuclease/helicase + flap endonuclease) along with the end-joining ligase(s). Suppression of end-edited joining on lowering the reaction temperature to 17 degrees or 14 degrees C, despite efficient faithful joining, indicated that this enzymatic activity is retarded at low temperature. Though the efficiency and fidelity of joining were termini-dependent, the orientation of joining was random. Lack of preference for homologous ends (H:H or T:T), as well as efficient joining of heterologous DNA (pUC12/pBR322) having two different blunt termini, showed that the end joining could occur independent of extensive/terminal homology. Retention of radioactivity on end joining of (alpha-32P)dCTP end-filled cohesive termini, and lack of their junctional cleavability, apparently due to restriction site duplication, suggested direct double strand ligation. Thus it is demonstrated that mouse male germ cells possess an efficient DNA end-joining activity, involving either a major pathway of precise joining, or a minor end-deleted joining, and it seems to be achieved by a multienzymatic complex as suggested also for somatic cells by others. These results show that mammalian male germ cells that are proficient in homologous recombination utilize nonhomologous end-joining (NHEJ) mechanism for DSB processing and therefore NHEJ is a conserved, universal pathway for the vital function of DSB repair.
Department of Zoology, Banaras Hindu University, Varanasi, India.
C R
CR
Sathees
M J
MJ
Raman
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002457:Cell Extracts / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D005854:Germ Cells / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D012150:Polymorphism, Restriction Fragment Length; D013696:Temperature; D013737:Testis / Q000378:metabolism*
D002457:Cell Extracts; D000255:Adenosine Triphosphate; D004247:DNA; D004720:Endonucleases; D005092:Exonucleases; D004262:DNA Restriction Enzymes; D011088:DNA Ligases; D008274:Magnesium