Excision Repair Protein Radl
نویسندگان
چکیده
Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cereuisiae, many genes required to repair DNA doublestrand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cereuisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cereuisiae excision repair protein Radl, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cereuisiae, in that it requires an NER protein. The biochemical properties of the Radl protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion. H OMOLOGOUS recombination is an essential feature of meiosis in many organisms. Recombination ensures the accurate disjunction of homologous chromosomes from one another by allowing the formation of physical linkages (chiasmata) derived from reciprocal exchange events (HAWLEY 1988). The molecular pathway by which recombination occurs is unknown, although a number of attractive models have been proposed (for review, see STAHL 1994). The model proposed by HOLLIDAY (1964) 30 years ago has been particularly influential, contributing two key features that have been incorporated into all subsequent models. The first of these is the creation of heteroduplex DNA, in which each strand of a double-stranded DNA helix is derived from a different parental molecule, as a central component of the recombination process. The existence of heteroduplex DNA has been confirmed by both physical studies (GOYON and LICHTEN 1993; NAG and PETES 1993) and the observation of postmeiotic segregation (PMS) events (WHITE et al. 1985). PMS occurs when a mismatch within heteroduplex DNA is not. repaired through meiosis and both sequences become fixed in the first postmeiotic round of DNA synthesis. Usually, however, mismatches within heteroduplex are repaired, thereby either restoring the sequence originally on that chromatid or replacing it with the sequence of the homologous chromatid. The latter possibility results in gene conversion, the nonreciprocal transfer of information from one site to another. Corresponding author: R. Scott Hawley, Section of Molecular and Cellular Biology, University of California, Davis, CA 95616. E-mail: [email protected] Genetics 141: 619-627 ( Octoher, 1995) The second important feature of HOLLIDAY’S model is the Holliday junction, a chi-shaped DNA structure connecting two parental DNA molecules. Resolution of a Holliday junction occurs when two strands of like polarity are cleaved, and their ends interchanged and religated. Depending on the two strands chosen, resolution can result in a crossover (i .e. , the exchange of flanking markers) or a noncrossover. Because proposed recombination intermediates contain one or two Hollidayjunctions adjacent to or flanking a region of heteroduplex DNA, gene conversion or PMS can be associated with both crossovers and noncrossovers. Clues to the molecular mechanism of meiotic recombination come from the observation that many of the genes required for this process are also required to repair certain types of DNA damage. In the yeast Succharomyces cerevisiae, a number of meiotic recombination genes are also required to repair DNA double-strand breaks (GAME et al. 1980; PRAKASH et al. 1980), suggesting models in which recombination is initiated by a double-strand break (SZOSTAK et al. 1983). In contrast, mutations in genes required for the nucleotide excision repair (NER) pathway, a versatile system that repairs many types of DNA damage (for review, see HOEIJMAKERS 1993; TANAKA and WOOD 1994; FREIDBERG et al. 1995), have no apparent effects on meiotic recombination in S. cerevisiae (SNOW 1968; PRAKASH et al. 1993). Many of the genes known to be required for meiotic recombination in Drosophila melanogaster are also required in mitotic cells (BAKER et al. 1978). Unlike the case in S. cerevisiae, however, at least one of these, mei9, is required for nucleotide excision repair (BOYD et 620 J. J. Sekelsky et al. al. 1976b; HARRIS and BOYD 1980). Mutations in mei-9 were first recovered in a screen by BAKER and CARPENTER (1972) for X-linked mutations causing high levels of meiotic nondisjunction. Meiotic nondisjunction in females homozygous for mei-9 mutations results from a decrease in the level of meiotic crossing over to < lo% of the normal level. Despite the substantial decrease in reciprocal exchange, meiotic gene conversion occurs at an approximately normal level (ROMANS 1980; CARPENTER 1982). However, mei-9 females exhibit high levels of PMS, which is manifested in the progeny as individuals who carry a single maternal chromosome but are mosaic for both maternal alleles (ROMANS 1980; CARPENTER 1982). Hence, m'-9 females are capable of generating recombination intermediates containing heteroduplex DNA but are defective both in the repair of mismatches within the heteroduplex and in the resolution of these intermediates as reciprocal exchanges. Alleles of mei-9 have also been recovered in screens for mutations conferring sensitivity to mutagens (BOYD et al. 1976a). At least some of the mutagen sensitivity of mei-9 mutants stems from an absolute block in NER (BOYD et al. 1976b; HARRIS and BOYD 1980). The requirement for an NER gene in meiotic exchange is somewhat surprising, given that no meiotic recombination role has been found for any NER gene in S. cermisiae. To understand the role of mei-9in meiotic recombination, we analyzed the gene at the molecular level. We found that mei-9 encodes a homologue of the yeast excision repair protein Radl, which is not required for meiotic recombination in S. cermisiae. MATERIALS AND METHODS Drosophila stocks and culture: Except where noted, genetic markers are described in LINDSLEY and ZIMM (1992) and FLYBASE (1994). Flies were reared on standard cornmealmolassesdextrose medium at 25". Methyl methanesulfonate treatment: To test for sensitivity to methyl methanesulfonate (MMS), adults were crossed in glass shell vials at 25" for 2 days before being removed. After one additional day, 250 p10.08-0.1% MMS (Sigma) in water was added to the medium. Pelement construct and transformation: P{w' mei-9+] was created by subcloning sequences from the EcoRI site in XXIII.62 (PFLUGFELDER et al. 1990), which is immediately distal to a genomic BamHI site, to the NotI site within the transcription unit proximal to mei-9, into pCaSpeR4 (PIRROnA 1988). Germline transformation was carried out essentially as in RUBIN and SPRADLING (1982). To test for rescue of MMS sensitivity, single w; P{w' ma9+]/+ males were mated to three w mi-pT2 /FM7, B females. The progeny larvae were treated with MMS as described above, and the number of BC w+ and B+ wmales that eclosed were counted. Meiotic nondisjunction was measured by cAossing two to three w mei-pT2; P(w" mei-9f] females to XY, In(1)EN Y f B; C(4)RM, tit$ males. The normal progeny of this cross are w mei-pT2/XY, In(l)EN, Y f B (B females) and w ma-9'"/ 0 (B+ males). Half of the diplo-X ova are recovered as w mei -pT2 /w mi -9" (B+ females), whereas the other half die. Similarly, half of the nullo-X ova are recovered as X Y , In(l)EN, Y f B/O (B males), and half die. The Xnondisjunction (ND) frequency is corrected for the loss of half of the excep tional progeny. Location of the med9R" P element: The position of the mi-9R" P-element was determined by PCR. Reactions contained 10-20 ng genomic DNA, 200 pM each dNTP, 100 pmol of each primer (CGATTGATTGTATCTTCC, corresponding to mei-9 bases 839-822 on the reverse strand, and CCCGCGGCCGCGACGGGACCACCTTATGTTATM'CATC, which contains the P element 31-bp inverted repeat and a NotI site, kindly provided by BIUAN CALVI), 1 mM MgC12, and 2 U Taq DNA polymerase (Promega) in 50 p1 of 1 X buffer supplied. Reactions were subjected to 25 cycles of 94", then 55", then 72" for 1 min each. The resulting fragment, which was amplified from mei-yT' but not from either of two revertants or from either mi-9Rr2 or mei-9RT4, was digested with PstI and NotI and subcloned into pBlueScript KS+ (Stratagene) for sequencing. Sequencing and sequence analysis: Double-strand sequencing was done using the Sequenase version 2.0 kit ( U S Biochemicals), using subclones and gene-specific primers. DNA sequences were assembled and analyzed with the University of Wisconsin Genetics Computing Group (UWGCG) programs ( DEVEREUX et al. 1984). Searches of the sequence databases were done on the National Center for Biotechnology and Information (NCBI) network server using the Basic Local Alignment Search Tool (BLAST) (ALTSCHUL et al. 1990). Protein sequence alignments were generated with the UWGCG programs GAP and PILEUP, using the BLOSUM62 amino acid substitution matrix (HENIKOFF and HENIKOFF 1992). Genebank accession umber: The GenBank accession number for the mei-9 sequence reported for in this paper is U27181.
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