FIG. 2.

Nature of Uve1p-generated DNA strand scission products and activity of full-length Uve1p. (A) Analysis of 5′ termini of Uve1p-generated DNA cleavage products with *CX/AY-31mer. 3′-end-labeled oligonucleotide with C/A mismatch (sequence on bottom) was reacted with GΔ228-Uve1p and then further treated with PNK or CIP as indicated. Lane 1 represents buffer treatment only. X* indicates base mismatch site. Arrows a and b indicate sites of Uve1p cleavage. (B) Full-length Uve1p possesses mismatch endonuclease activity. 5′-end-labeled duplex *CX/AY-31mer was incubated with crude extracts of cells expressing either GFL-Uve1p (lane 1) or GΔ228-Uve1p (lane 2) and cells expressing the GST tag alone (lane 3) or with E. coli endonuclease (Endo) V, a known mismatch endonuclease (lane 4). Arrows indicate cleavage sites immediately (arrow a) and one nucleotide (arrow b) 5′ to the mismatch site. Arrow V indicates E. coli endonuclease V cleavage 3′ to the mismatch site and was used as a position reference. Bands below arrows (indicated by asterisks) correspond to shortened products due to a weak 3′-to-5′ exonuclease activity present in the Uve1p preparations (see text for details). Reaction products on DNA sequencing-type gels were analyzed as described for Fig. 1.