A Uve1p-mediated mismatch repair pathway in Schizosaccharomyces pombe

Abstract

UV damage endonuclease (Uve1p) from Schizosaccharomyces pombe was initially described as a DNA repair enzyme specific for the repair of UV light-induced photoproducts and proposed as the initial step in an alternative excision repair pathway. Here we present biochemical and genetic evidence demonstrating that Uve1p is also a mismatch repair endonuclease which recognizes and cleaves DNA 5' to the mispaired base in a strand-specific manner. The biochemical properties of the Uve1p-mediated mismatch endonuclease activity are similar to those of the Uve1p-mediated UV photoproduct endonuclease. Mutants lacking Uve1p display a spontaneous mutator phenotype, further confirming the notion that Uve1p plays a role in mismatch repair. These results suggest that Uve1p has a surprisingly broad substrate specificity and may function as a general type of DNA repair protein with the capacity to initiate mismatch repair in certain organisms.

FIG. 1

GΔ228-Uve1p recognizes 12 different base mismatch combinations. The 3′-end-labeled oligonucleotide series X/Y-31mer (sequence given at bottom; asterisk indicates labeled strand and labeled terminus) was used to assess Uve1p cleavage activity on 16 different base pair and base mispair combinations (Table 1). Base mispairs are indicated above numbered lanes, with asterisks denoting bases on the labeled strand for G series (A), A series (B), C series (C), and T series (D) treated with purified GΔ228-Uve1p (odd-numbered lanes) or mock reactions (even-numbered lanes). Reaction products were analyzed on DNA sequencing-type gels. Arrows indicate Uve1p cleavage sites immediately (arrow a) and one (arrow b) and two (arrow c) nucleotides 5′ to the mismatch site. G and C+T base-specific chemical cleavage DNA sequencing ladders were run in adjacent lanes as nucleotide position markers.

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.

FIG. 3

GΔ228-Uve1p requires divalent cations for mismatch recognition. 5′-end-labeled duplex *CX/AY-31mer was incubated with GΔ228-Uve1p (lanes 1 and 4) or buffer only (control [CON]; lanes 2 and 5) in the presence (lanes 1 and 2) or absence (lanes 4 and 5) of Mn²⁺. C* indicates base mismatch site. Arrows a and b indicate Uve1p cleavage positions. E. coli endonuclease (Endo) V-reacted oligonucleotide (arrow v, lane 3) and C+T and G+A sequencing ladders included as nucleotide position markers are marked. Bands below arrow b (indicated by asterisks) correspond to shortened products due to 3′-to-5′ exonuclease activity (described in the legend to Fig. 2).

FIG. 4

GΔ228-Uve1p mismatch endonuclease and GΔ228-Uve1p UV photoproduct endonuclease compete for the same substrates. GΔ228-Uve1p was incubated with 3′-end-labeled duplex *CX/AY-31mer (Table 1) in the presence of increasing amounts of unlabeled duplex CPD-30mer (squares), duplex GX/CY-31mer (triangles), or duplex CX/AY-31mer (circles). The Uve1p-mediated DNA cleavage products were analyzed on DNA sequencing gels, and the extent of strand scission was quantified by PhosphorImager analysis (Materials and Methods). Uve1p activity is expressed as percentage of the cleavage observed relative to that observed in the absence of any competitor (defined as 100% activity). The error bars indicate the mean ± standard deviation from three separate experiments.

FIG. 5

Uve1p incises only one strand of a duplex containing a base mismatch. (A) 3′-end-labeled *CX/AY-41mer was incubated with restriction enzyme DdeI (lane 1), GΔ228-Uve1p (lane 2), or buffer (lane 3). The reaction products were analyzed on a nondenaturing gel as described in the text for the presence of DNA double-strand break products (arrow dsb). Arrows b and c indicate the primary cleavage site for Uve1p on this substrate. (B) 3′-end-labeled *CX/AY-41mer or CX/*AY-41mer was incubated with GΔ228-Uve1p (+ lanes) or buffer (− lanes) and analyzed on denaturing, DNA sequencing-type gels as described in the text. Arrows b and c indicate positions of major Uve1p cleavage events relative to the mismatched base (asterisk) position. G+A and C+T base-specific sequencing ladders are included in outside lanes as nucleotide position markers.