We previously described a 5-3 exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5-3 exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair.The currently favored models for homologous recombination predict a requirement for nucleases at several steps in the recombination reaction (18,37,55). Endonucleases are envisioned to function as initiators of recombination and in the resolution of crossed-strand intermediates. Exonucleases are thought to process break sites to generate single-stranded DNA, the substrate for binding by homologous pairing proteins. Late steps in the reaction, such as the repair of heteroduplex DNA (mismatch correction), also require the activity of exonucleases. The importance of exonucleases in recombination has been clearly demonstrated in Escherichia coli, in which 5Ј-to-3Ј exonucleases are required for all homologous recombination pathways (30). In addition, the single-stranded exonucleases RecJ (5Ј), exonuclease VII (Exo VII) (5Ј and 3Ј), and Exo I (3Ј) are required for mismatch repair in vitro (8).In vivo studies of double-strand break (DSB) repair in Saccharomyces cerevisiae provide further support for the role of exonucleases in recombination. After HO endonuclease cleavage at the MAT locus during mating-type switching, the DNA is resected to produce a 3Ј tail on the distal side of the HO cut site (64). This single-strand tail is believed to invade the donor locus, thus initiating the transfer of information. Formation of the 3Ј single-stranded tail is thought to result from the activity of a double-stranded 5Ј-3Ј exonuclease, but it could arise from the combined activities of a helicase and single-stranded endonuclease or exonuclease (35). Although mutation of several known recombination genes prevents mating-type switching, none appears to block the exonucleolytic processing step (49, 64). However, the degradation of the 5Ј strand is slower in rad50 and xrs2 mutants, suggesting that these genes may encode or regulate the activity of a nuclease (21). Most meiotic recombination hot spots are cleaved by endonucleases during meiosis, and the resulting DSBs are ...

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggests that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.In eucaryotes, the transcription units which direct the synthesis of the rRNA precursor (pre-rRNA), eventually processed into the mature rRNA species, are separated by long intergenic segments originally called nontranscribed spacers (NTS) on the basis of electron microscopy observations (27). Sequences sufficient to drive faithful RNA polymerase I (Pol I)-dependent initiation are included within the NTS portion immediately preceding the pre-rRNA start site (29, 38). In many species, however, functional assays have shown that additional upstream NTS elements significantly influence the rate of pre-rRNA transcription initiation (29,38). In Xenopus short sequences, the 60/81-bp elements, clustered in tandem within the NTS, stimulate transcription from the pre-rRNA promoter when placed in either orientation and at variable distance, strictly reminiscent in their mode of action of polymerase II enhancers (19,34). Pol I enhancers have also been identified in Saccharomyces cerevisiae (11,12,18,25) and rat (9) ribosomal DNA (rDNA) spacers. The NTS of many species also contain one or more Pol I-dependent sites of transcription initiation, in most cases related in sequence to the DNA regions surrounding the pre-rRNA transcription start site (3,21,23,28,39,41); spacer promoters are either interspersed (3, 29) or juxtaposed (18, 25) with enhancerlike elements.Arrays of spacer promoters are a physical hallmark of the Drosophila rDNA spacers (26,30,37,40). The Drosophila melanogaster rDNA NTS is composed of three adjacent regions which are internally repetitious, in turn composed of 95-, 330-, and 24...

No abstract

In Drosophila melanogaster, 240-base-pair (bp) repeats, clustered in tandem arrays within the ribosomal DNA nontranscribed spacer region, include sites of RNA polymerase I-dependent transcription initiation and elements that stimulate the rate of transcription from the downstream precursor rRNA (pre-rRNA) promoter. We have analyzed the in vivo transcriptional activity of a large set of recombinant constructs in which tandem arrays of distinct segments derived from a 240-bp repeat were inserted upstream of the pre-rRNA promoter. The results indicate that activating spacer elements are confined to a region of 70 bp. Enhancing units overlap with spacer promoters, since DNA segments that stimulate transcription at the gene promoter also efficiently drive transcription initiation. The finding that artificial spacer arrays invariably stimulate pre-rRNA transcription initiation in an orientation-dependent fashion suggest that spacer-initiated transcription is involved in the enhancement process. The minimal spacer activating segment includes a perfect copy of a core domain of the gene promoter extending from -24 to +10 flanked by poorly homologous upstream DNA sequences. Spacer and gene promoters are functionally interchangeable as activating units. However, the different combination of DNA elements within the two determines a functional hierarchy, as only the pre-rRNA promoter is responsive to the stimulatory action of upstream units.

No abstract