DNA STRAND BREAKAGE DURING PHYSIOLOGICAL APOPTOSIS OF THE EMBRYONIC CHICK LENS - FREE 3' OH END SINGLE-STRAND BREAKS DO NOT ACCUMULATE EVENIN THE PRESENCE OF A CATION-INDEPENDENT DEOXYRIBONUCLEASE

Epithelial cells from the lens equator differentiate into elongated fi ber cells. In the final steps of differentiation, the chromatin appear s quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at l east two polypeptides of 30 and 40 kDa have been identified. To identi fy the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in f iber and epithelial cells from chick embryonic tenses. There is no acc umulation of single-strand breaks (SSB) with 3'OH ends in lens fiber c ells during embryonic development. Such damage can be increased in the se cells by treatment with DNAase I indicating the absence of an inhib itor of the nick translation reaction in fiber cells. However, there a re indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of brea ks is also indicated by the large amounts of (ADP-ribose), found in le ns fibers particularly at 11 days of embryonic development (E11) as AD P-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentat ion only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiologica l role. Long incubations produce larger numbers of SSB in epithelia th an fibers. The SSB in the fibers may have been converted into double-s trand breaks (D SB), seen as nucleosomal fragments, and therefore no l onger act as substrates for nick translation. The nuclease activity re sponsible for SSB production is independent of divalent cations and co uld be implicated in lens terminal differentiation.