Joining of DNA ends bearing non-matching 3'-overhangs.

Using the Xenopus egg extract as an in vitro system for double strand break repair, the joining of DNA ends bearing non-complementary, homopolymeric, 4nt 3'-protruding single strands ("overhangs") was examined. Such 3'-overhangs can not be filled-in and cannot align and anneal by canonical base pair interactions, thus presenting a special challenge to the repair machinery. The results indicate that two such non-matching 3'-overhangs typically overlap by 2nt forming non-canonical base pairs, from which the filling-in of the remaining gaps is primed. The repair reaction is inhibited in Ku-depleted extracts. Unexpectedly, with some of the substrates the predominant repair products were joints with no nucleotide loss, suggesting that the two DNA ends aligned without overlap. However, the additional finding that an activity in the egg extract adds one or a few nucleotides to a fraction of the 3'-ends favors a model in which most or all of the zero-loss joints are the net result of 3'-overhang extension and 2bp overlap formation. The nucleotide addition reaction is stimulated by increasing the concentration of the complementary dNTP or ddNTP in the extract, suggesting a process templated by free nucleotides and the involvement of a DNA polymerase-like activity.