CHARACTERIZATION OF DNA END JOINING IN A MAMMALIAN-CELL NUCLEAR EXTRACT - JUNCTION FORMATION IS ACCOMPANIED BY NUCLEOTIDE LOSS, WHICH IS LIMITED AND UNIFORM BUT NOT SITE-SPECIFIC

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this p rocess are largely unknown. A previous report has described an activit y from mammalian cell nuclei that is capable of multimerizing blunt-en ded DNA substrates (R. Fishel, M. K. Derbyshire, S. P. Moore, and C. S . H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferential ly join linear plasmid substrates in either a head-to-head or tail-to- tail configuration, that the joining reaction is covalent, and that th e joining is accompanied by loss of sequence at the junction. Sequenci ng revealed that there was a loss of a uniform number of nucleotides f rom junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nu cleotides lost was affected by the precise terminal sequence. There wa s no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Us ing a pair of plasmid molecules with distinguishable restriction enzym e sites, we also observed that blunt-ended DNA substrates could join w ith those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was a uniform loss of nuc leotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located tow ard their ends. Although either model can explain the preferential for mation of head-to-head and tail-to-tail products, the latter predicts the precise loss of nucleotides observed. These activities are found i n all cell lines examined so far and most likely represent an importan t repair activity of the mammalian cell.