HOPl Gene: Evidence for Multimeric Assembly in Meiosis

The HOPl gene of Saccharomyces cereuisiae has been shown to play an important role in meiotic synapsis. In this study we analyzed the mechanism of this function by phenotypic characterization of novel in-frame linker-insertion mutations located at various sites throughout the HOPl coding sequence. Among 12 mutations found to cause defects in meiotic recombination and spore viability, three were temperature-sensitive for the spore viability defect. Although substantial meiotic recombination was found for these conditional alleles at the restrictive temperature, the level of exchange measured in spo13 meiosis was reduced in some of the monitored intervals, indicating that nondisjunction resulting from a deficit in crossing over could account for SP013 spore inviability. Intragenic complementation between linker-insertion alleles was assessed by testing the viability of spores generated from heteroallelic diploids after SP013 meiosis. Complex patterns of complementation and enhancement of the spore-inviability phenotype indicate that HOPl functions in a multimeric complex. In addition, the ability of alleles which map near the carboxyl terminus to complement several other alleles provides evidence for a functional domain in this region of the protein. Two previously identified multicopy suppressors of the conditional hopl-628" allele were tested for their effects in cells bearing the linker-insertion hopl alleles. Overexpression of R E C m from a 2p plasmid was shown to enhance the spore viability of every allele tested, including a hopl disruption allele. On the other hand, suppression by overexpression of REDl from a 2p plasmid was found only for allele hopl-628b. Surprisingly, similar overexpression of REDl in strains bearing several other conditional hop1 linker-insertion alleles caused enhanced spore lethality. This finding, in conjunction with the evidence for a carboxy-terminal domain, provides new insight into the nature of interactions between the HOPl and REDl products in meiosis. M EIOSIS typically involves two successive rounds of nuclear division that are coupled in a manner that yields four haploid progeny from the initial diploid cell. During the first of these divisions, the homologous centromeres segregate to opposite poles of the meiotic spindle while sister centromeres remain associated with one another. The efficacy of this reductional segregation depends on recombination between the homologs to create the chiasmata that are crucial for chromosomal alignment on the spindle during the first meiotic division (NICKLAS 1967). In most eukaryotes, an important element in meiotic prophase is the synaptonemal complex (SC), which serves as a central scaffold between the paired homologs during the time of crossing over (reviewed in VON WETTSTEIN et a l . 1984). Sites of crossing over are marked by recombination nodules (CARPENTER 1979) and specific modifications of the SC are later retained within the chiasmata (VON WETTSTEIN et a l . 1984). When formation of the SC and/or crossing over per ' Present address: Department of Biochemistry, SJ-70, University of Wash* To whom correspondence should be addressed. ington, Seattle, Washington 98195. Genetics 136 449-464 (February, 1994) se are blocked by relevant mutations, the deficiency for chiasmata prevents the homologs from adhering properly to one another at metaphase I and the resulting aneuploidy is often lethal to the progeny (reviewed in BAKER et al. 1976). Specific mechanisms of SC function in meiosis have begun to be clarified by a variety of genetic and cytological approaches. Recent evidence by ENGEBRECHT et a l . (1 990) in Saccharomyces cerevisiae suggests that the SC is important for promoting the specific type of meiotic exchange that is required for proper homolog segregation, since the low level of reciprocal exchange occurring in a mer1 mutant strain can be made more effective in promoting proper meiosis I disjunction when SC is present. In addition, kinetic analysis of sporulation in S. cerevisiae demonstrates that both the formation of double-strand breaks (PADMORE et a l . 1991) and the alignment of chromosome regional-specific probes (SCHERTHAN et al. 1992) occur prior to tripartite SC formation, suggesting that the requirement for fully formed SC arises late in meiotic prophase. Among the specific functions that may be attributed to the SC is chiasma 450 D. B. Friedman, N. M. Hollingsworth and B. Byers interference, since patterns of multiple exchanges are not subject to interference in Schizosaccharomyces pombe and Aspergillus nidulans, both of which lack SC [S. pombe: BAHLER et al. (1993), OLSON et a l . (1978) and SNOW (1979); A. nidulans: EGEL-MITANI et a l . ( 1 982) and STRICKLAND (1958)l. There are many genes in S. cerevisiae that are essential for the production of viable euploid spores, and several of these genes seem to be directly involved in the recombination process. Spore death in strains mutant for HOPl or R E D l , on the other hand, is believed to result from perturbations in homolog pairing, as these strains remain proficient for recombination between duplicated chromosomal elements (HOLLINGSWORTH and BYERS 1989; ROCKMILL and ROEDER 1990). Furthermore, epistasis analysis indicates that the products of these genes, and perhaps the more recently characterized gene MEKl (ROCKMILL and ROEDER 1991), act together in a common function that promotes some aspect of recombination between homologs (ROCKMILL and ROEDER 1990). Consistent with the idea that the relevant meiotic function of these genes is distinct from other functions needed at an earlier stage of meiosis, a large screen for mutations suppressing rad52 lethality yielded new alleles of several genes that affect early functions, but none in HOPl or REDl (MALONE et al. 1991). The 605-amino acid HOPl protein contains a cysteine-rich region which may contain a single nonclassical zinc-finger DNA binding motif, and one cysteine residue in this region has been shown to be necessary for full HOPl activity (HOLLINGSWORTH et al. 1990). Atomic mass spectroscopy of purified HOPl protein reveals that the protein contains 1-2 mol Zn2+/mole protein while a mutant protein lacking one of the conserved cysteine residues does not (N. HOLLINGSWORTH and A. JOHNSON, unpublished results). The HOPl protein exhibits nonspecific DNA binding activity (unpublished observations) and this activity is Zn2+-dependent (K. MUNIYAPPA, personal communication). Immunochemical methods reveal that HOPl is associated with the synapsed meiotic chromosomes (HOLLINGSWORTH et a l . 1990), being localized to a continuous central region that is narrower than the overall bivalent (F. KLEIN, personal communication). These findings strongly suggest that the HOPl protein plays a important structural role in the meiotic bivalent, probably serving as a component of the synaptonemal complex. We present here the characterization of 16 in-frame linker-insertion mutations located at specific sites throughout the HOPl open reading frame. Although some of these alleles permit moderate levels of recombination among the rare viable spores produced when SP013 is present, spol3 meiosis reveals that most cells are markedly deficient in recombination within some TABLE 1 S. cerezrisiae strains