Bioinformatic Analyses Implicate the Collaborating Meiotic Crossover͞chiasma Proteins Zip2, Zip3, and Spo22͞zip4 in Ubiquitin Labeling

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Bioinformatic analyses implicate the collaborating meiotic crossover͞chiasma proteins Zip2, Zip3, and Spo22͞Zip4 in ubiquitin labeling

Jason Perry*, Nancy Kleckner*†, and G. Valentin Bo¨ rner‡

*Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; and ‡Biological, Geological, and Environmental Sciences Department, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115

Contributed by Nancy Kleckner, October 3, 2005 Zip2 and Zip3 are meiosis-specific proteins that, in collaboration uration into crossovers are subject to a specifically programmed with several partners, act at the sites of crossover-designated, progression block, which is then overcome to permit formation of axis-associated recombinational interactions to mediate cross- SEIs and nucleation of SCs. Progression through this block requires over͞chiasma formation. Here, Spo22 (also called Zip4) is identified the coordinate action of a particular set of proteins, the ZMM as a probable functional collaborator of Zip2͞3. The molecular roles group, which then also mediates the ensuing DNA and structural of Zip2, Zip3, and Spo22͞Zip4 are unknown. All three proteins are outcomes (2). Members of the ZMM group identified thus far are part of a small evolutionary cohort comprising similar homologs in Mer3, Msh4͞5, Zip1, Zip2, and Zip3 (2). The entire ZMM ensem- four related yeasts. Zip3 is shown to contain a RING finger whose ble likely acts directly at the sites of developing crossovers͞ structural features most closely match those of known ubiquitin chiasmata, at this and later stages, as indicated by co- E3s. Further, Zip3 exhibits major domainal homologies to Rad18, a immunoprecipitation, two-hybrid interactions, and known DNA-binding ubiquitin E3. Also described is an approach to immunocytological studies (5, 6). the identification and mapping of repeated protein sequence Biochemical functions can be assigned for only some of the ZMM motifs, Alignment Based Repeat Annotation (ABRA), that we have proteins: Mer3 is a DNA helicase, Msh4͞5 is a meiosis-specific developed. When ABRA is applied to Zip2 and Spo22͞Zip4, they MutS homolog that does not mediate mismatch repair, and Zip1 is emerge as a 14-blade WD40-like repeat protein and a 22-unit the transverse filament component of the SC (reviewed in refs. 2 tetratricopeptide repeat protein, respectively. WD40 repeats of and 7). There is almost no information regarding the molecular Cdc20, Cdh1, and Cdc16 and tetratricopeptide repeats of Cdc16, functions of Zip2 or Zip3. Cdc23, and Cdc27, all components of the anaphase-promoting Here we identify Spo22 as an additional member of the ZMM complex, are also analyzed. These and other findings suggest that group. Spo22 has also been studied by the Roeder laboratory, who Zip2, Zip3, and Zip4 act together to mediate a process that involves have renamed it Zip4 (G. S. Roeder, personal communication). We Zip3-mediated ubiquitin labeling, potentially as a unique type of also present bioinformatic analyses that, in combination with other ubiquitin-conjugating complex. information, lead to the proposal that Zip2, Zip3, and Spo22͞Zip4 are components of a meiosis-specific complex for protein labeling. bioinformatics ͉ meiosis ͉ recombination ͉ signaling ͉ genetics Available evidence points to ubiquitin as the ligated moiety, although SUMO (small ubiquitin-like modifier) cannot be rigorously prominent feature of meiosis is a programmed progression of excluded as a possibility. The presented analyses depend in part Alocal interhomolog interactions that culminates in formation upon Alignment Based Repeat Annotation (ABRA), an approach of DNA crossovers and their cytological counterparts, chiasmata. to the identification and mapping of repeated sequence motifs within a set of related but evolutionarily diverged proteins. ABRA Recombination is initiated by the occurrence of a large number of ͞ programmed double-strand breaks (DSBs) (1). Only a few of these is described and then applied to Zip2, Spo22 Zip4, and structurally ͞ related components of the anaphase-promoting complex (APC). A DSBs mature into crossovers chiasmata. The remaining majority ͞ ͞ interact with a homolog partner but ultimately are resolved without model for Zip2 3 4 as a unique ubiquitin ligation complex is any accompanying exchange, i.e., as ‘‘noncrossovers.’’ discussed. The decision as to which recombinational interactions will ma- Materials and Methods ture as crossovers and which will mature as noncrossovers appears to be made at late leptotene of meiotic prophase and to trigger A transposon insertion library (8) was screened by using the ͞ homothallic ste7-ts strain NKY3643 (HO͞HO, MATa͞MAT␣, ste7- chromosomal progression through the ensuing ‘‘leptotene ͞ ͞ ͞ ͞ zygotene’’ transition (2). At the time this decision is made, DSBs 1 ste7-1, ura3 ura3, leu2 leu2, ade2::LK ade2::LK) by the method- have occurred and have probably begun to interact with their ology of McKee and Kleckner (9): A population of haploid cells was partner duplexes; concomitantly, at the chromosome structure transformed and plated under selective conditions that also permit level, axes have formed and homologs are linked by bridge struc- self-diploidization. Resulting diploid colonies were screened for tures that support corresponding recombination complexes. Once spore formation at 23°C and 33°C by replica plating to sporulation medium and visualization of UV fluorescence (2). Fig. 1A used a particular recombinational ensemble has been designated to spore clones of homothallic strain NKY3644, which is NKY3643 become a crossover, further progression occurs at both levels. At (above) also carrying spo11(Y135F)-HA-URA3͞SPO11, the DNA level, the nascent DSB͞partner interaction progresses to spo22::Hyg4MX͞SPO22. Fig. 1 B–D used NKY3639 (wild type) and an intermediate involving stable exchange of strands between the interacting duplexes at one end of the break (the ‘‘single-end

invasion’’ or SEI) (3). At the structural level, bridges disappear, Conflict of interest statement: No conflicts declared. accompanied by nucleation of the synaptonemal complex (SC), Abbreviations: ABRA, Alignment Based Repeat Annotation; APC, anaphase-promoting which then polymerizes outward from these sites. Fully formed SC complex; DSB, double-strand break; SC, synaptonemal complex; SUMO, small ubiquitin-like is a close-packed array of transverse filaments that links chromo- modifier; TPR, tetratricopeptide repeat. some axes along their entire lengths (reviewed in ref. 4). †To whom correspondence should be addressed at: Harvard University, 7 Divinity Avenue, Molecular genetic studies have shown that DSB-containing Room 301, Cambridge, MA 02138. E-mail: [email protected]. recombinational interaction complexes designated for future mat- © 2005 by The National Academy of Sciences of the USA

17594–17599 ͉ PNAS ͉ December 6, 2005 ͉ vol. 102 ͉ no. 49 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508581102 Downloaded by guest on September 24, 2021 protein database (accessed at www.ncbi.nlm.nih.gov; cutoff threshold E ϭ 0.005) was carried out by using the sequence from S. cerevisiae as the query (11). This search effectively converges after the second iteration upon retrieving obvious homologs from K. lactis, C. glabrata, and C. albicans (partial sequence) plus other sequences exhibiting less strong homology. An analogous search for Zip2s converges regularly after the second iteration, in this case yielding only homologous sequences from the same three yeasts identified as having related Zip3s. A search for Spo22͞Zip4s identified homologs from these same three organisms in the first iteration (E Յ 3eϪ31); however, this search is perpetuated by the inclusion of functionally unrelated tetratricopeptide repeat (TPR)- containing proteins (below).

Zip3s Contain a RING Finger Motif. Pfam database searches are designed to detect known protein sequence motifs by trained hidden Markov models (12). Pfam analysis of Zip3s gives no significant information (accessed through http:͞͞pfam.wustl.edu͞ hmmsearch.shtml; E ϭ 1.0, a default that will report Ϸ1 false positive hit per query sequence). However, a PSI-BLAST search with amino acid residues 1–91 of S. cerevisiae Zip3, when carried to the third iteration, identified divergent sequences with conserved potential metal ligand positions. When these sequences were analyzed by Pfam, they emerged as statistically significant (e.g., Arabidopsis thaliana CAB46003, E ϭ 5.8eϪ06) binuclear Zn RING (really interesting new gene) fingers. All functionally characterized RING fingers described to date

are E3 ubiquitin ligase modules, meaning that they transfer ubiq- BIOCHEMISTRY uitin from its activated form on an E2 molecule to a cognate target protein. MIZ (Msx-interacting zinc finger) domains are functionally analogous to ubiquitin E3s except that they can mediate the transfer Fig. 1. Characterization of spo22⌬͞zip4⌬.(A) Spore formation as visualized of SUMO from Ubc9 onto a target molecule. These and other types by UV ﬂuorescence of cell patches replica plated onto sporulation medium. of evolutionarily related Zn finger motifs (e.g., PHD and FYVE) spo22⌬ shows temperature- and SPO11-dependent failure in spore formation. are distinguishable from one another by specific ligand position (B–D) Events of meiosis in cultures undergoing synchronous meiosis, exactly as signatures (available at the Pfam server noted above). In the RING previously described (2). (B) Completion of the ﬁrst meiotic division (MI) as ubiquitin E3s analyzed thus far, the canonical ligand signature is indicated by appearance of two chromosome masses. (C and D) Appearance of LXXLXnLXLXXLXXLXnLXXL. The Zip3s of S. cerevisiae, K. crossover (CR) and noncrossover (NCR) recombination products at the lactis, and C. glabrata all contain exactly this arrangement as HIS4LEU2 hot spot. Gels in C are quantitated in D, which also includes data for illustrated by comparison with two known RING ubiquitin E3s, a previously analyzed zmm mutant (zip2⌬) taken from ref. 2. Rad18 and Apc11 (Fig. 2A). This is also the case with the likely Zip3 homolog in C. elegans (below). In contrast, we have examined over an isogenic derivative carrying a complete deletion of SPO22 100 MIZ-SUMO E3 domains, and none exhibits the ligand pattern (NKY3645) exactly as described (2). observed in ubiquitin E3s and Zip3s, consistent with its annotation as a different type of domain. Most, including experimentally Results verified MIZ-SUMO E3s from S. cerevisiae (Mms21) and Arabi- Identification of Spo22 as a Member of the ZMM Group. Mutants dopsis (Siz1, At5g60410), do not even contain the same number of lacking a ZMM protein exhibit specific temperature-modulated potential His or Cys ligands (13, 14). These indicators strongly meiotic phenotypes. At higher temperatures: reduced formation of suggest that Zip3s are ubiquitin E3s, although SUMO E3 ligase crossover recombination products (Ϸ15% of the wild-type level), activity cannot be formally excluded without experimentation. normal formation of noncrossover recombination products, and Spo11-dependent arrest at meiotic prophase, with failure to form Zip3s Share N- and C-Terminal Domainal Homology with Rad18, a DNA spores. At lower temperatures: crossover levels are reduced, non- Binding E3 That Promotes Mitotic DNA Break Repair. Zip3 is a crossover levels are increased, and exit from meiotic prophase and chromosome-associated protein (5, 6). The presence of an N- ensuing spore formation occur efficiently but with a delay (2). terminal RING finger occurring in a chromosomal protein is Additional ZMM genes were sought by introducing a library of reminiscent of another, more fully characterized S. cerevisiae pro- transposon insertion mutations into our standard strain background tein, Rad18. Rad18 is an ubiquitin-ligating E3 that heterodimerizes and screening for insertions that conferred a temperature- via its C terminus with the ubiquitin E2 enzyme Rad6 to promote dependent defect in spore formation. (Materials and Methods) One postreplicative DNA repair by monoubiquitinylating DNA pol30 such insertion was shown by genomic sequencing to lie in a (PCNA) (15, and references cited therein). To explore potential previously identified (10) meiosis-specific gene, SPO22. A strain relationships between these two proteins, we performed statistical carrying a complete deletion null mutation of this gene, spo22⌬, was T-COFFEE analysis of Zip3 in relation to Rad18 (16). Two regions constructed and found to exhibit the entire array of diagnostic zmm of high homology emerge: (i) the N-terminal RING finger domain mutant phenotypes (Fig. 1). Spo22 also emerged recently in another and (ii) a stretch of Ϸ70 C-terminal amino acids. Secondary mutant screen and was named Zip4 (above). structure analysis (Jpred; www.compbio.dundee.ac.uk͞ϳwww- jpred͞) further reveals structural homology throughout the C Saccharomyces cerevisiae Zip2, Zip3, and Spo22͞Zip4 Have Homologs termini of these proteins and permits final manual refinement of in Kluyveromyces lactis, Candida glabrata, and Candida albicans. A the T-COFFEE alignments (Fig. 2B). Analogous homologies also PSI-BLAST search for Zip3-related sequences in the nonredundant occur between the Zip3s and Rad18s of K. lactis and C. glabrata (not

Perry et al. PNAS ͉ December 6, 2005 ͉ vol. 102 ͉ no. 49 ͉ 17595 Downloaded by guest on September 24, 2021 analogous function, with Rad6 or another as-yet-to-be-identified E2. Comparative domain architecture diagrams are provided in Fig. 2C.

Development of ABRA and Its Application to Zip2s. Pfam database searches using any of the four retrieved Zip2 sequences as the query fail to identify statistically significant (E ϭ 1.0) domain signatures; automated T-COFFEE alignment analysis finds only Ϸ3% identity among the set of putative orthologs, and none of the tertiary structure algorithms linked to the Polish metaserver (http:͞͞ bioinfo.pl͞meta) were able to model full-length Zip2s with any reasonable degree of statistical significance (18). However, RA- DAR (rapid automatic detection and alignment of repeats) analysis (www.ebi.ac.uk͞Radar͞) suggested the presence, in all four sequences, of repetitive elements that are between 35 and 50 aa in length (19). We could not identify these segments as corresponding to any known repeat motif by PSI-BLAST, SMART, Pfam, or other profile search algorithms. However, visual inspection of the sequences indicated the presence of periodic aromatic amino acid residues. Moreover, automated T-COFFEE analysis revealed the presence of a single particularly conserved aliphatic-K-hydrophilic- L-X-W-D͞N motif near the center of the alignment. These two Fig. 2. Informatic analysis of Zip3s. (A) The RING ﬁngers of Zip3s compared sequence features are characteristic of WD40-like repeats. with those of Rad18s and Apc11s: Sc, S. cerevisiae; Cg, C. glabrata; Kl, K. lactis. To further explore this suggestion, we developed an approach The presumed zinc ligands are highlighted. (B) A second domain of homology that is generally applicable to the identification of any type of between Zip3s and Rad18s. Bars are helices and arrows above the sequences repeated structural motifs in a set of homologous but significantly are extended strands as determined by Jpred. Arrows below the sequences delimit the region of Rad18 that is necessary and sufﬁcient for heterodimer- divergent proteins: ABRA. ABRA analysis begins with a basic ization with the E2 Rad6 (see text). The boxed segment of ScZip3 (LKSD) was multiple sequence alignment, generated by T-COFFEE (Fig. 3 Top). determined by SUMOplot (www.abgent.com͞doc͞sumoplot) to be a high This basic alignment is then optimized manually by taking into probability SUMOylation target (91͞100). This feature is present only in the S. account three parameters: (i) visual inspection of a set of all cerevisiae sequence. (C) Comparison of the domain architectures of ScZip3 and pairwise alignments with respect to sequence identities; (ii) amino ScRad18. The diagram is approximately to scale. acid composition͞position analysis (e.g., aliphatic hydrophobic in position 1, H-bond acceptor in position 2, etc.); and (iii) secondary structure predictions, producing a refined alignment (Fig. 3, Mid- shown). In Rad18s, the C-terminal Zip3-related region comprises dle). In this refined alignment, structural elements and repeating the Rad6 (E2) association interface; moreover, a segment com- segments among various sequences are often immediately visible posed of two short strands followed by an amphipathic helix (Fig. (e.g., Fig. 3 Bottom). However, if they are not, the RADAR 2B) is necessary and sufficient for Rad18͞Rad6 heterodimerization algorithm can be useful by nucleating the repeat array in various (17). In Zip3s, this domain would presumptively perform an positions by identifying repetitive elements in individual sequences

Fig. 3. ABRA methodology.

17596 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508581102 Perry et al. Downloaded by guest on September 24, 2021 BIOCHEMISTRY

Fig. 4. Application of ABRA to ␤-repeat proteins. (A) Results of ABRA analysis of Zip2, Cdc20, and Cdh1 from S. cerevisiae. The bottom set of sequences is a Conserved Domain Database-derived structure based alignment of random WD40 repeats, shown for comparison. Zip2 emerges as a 14-blade WD40-like repeat protein. (B) Phylogenetic analysis of the repeats found in Zip2. The tree was constructed by using TreeTop. The numbers represent the percentage of times each branch topology was found during the bootstrap analysis. (C) Structural examples of 7-blade (Ski8, PDB ID code 1SQ9, Upper) and 14-blade (Unc78, PDB ID code 1NR0, Lower) WD40 proteins.

that are then inferred in all sequences in the alignment. Structural consistent with the paradigm that repeat proteins have arisen by motifs are then delimited by the fewest number of secondary successive tandem duplications. These results provide strong evi- structure elements (e.g., extended strands, ␣-helices) required to dence that the identified repeat units, which were defined and complete one iteration of the sequence repeat. These motifs are aligned to one another by sequence and secondary structure then aligned to one another for further repeat delimitation refine- elements, are in fact nonrandomly related. Interestingly, each unit ment. Once a repeating structural motif is identified by this has a tendency to be particularly similar to an adjacent unit, with approach, its specific nature is inferred by comparison with known defined pairs comprising units [2, 3], [4, 5], [9, 10], [11, 12], and [13, structural repeat motifs. 14]. This pattern could mean that single unit was duplicated first, With Zip2s, ABRA analysis reveals the presence of 14 reiterated followed by successive tandem duplications of the resulting pair of elements. Moreover, the lengths and the particular array of struc- units. tural and sequence features of these elements are most similar to Structural studies have shown that WD40 repeat units are those of WD40 repeat units (Fig. 4A and Figs. 6 and 7, which are arranged into a ␤-propeller. The last strand of the each repeat unit published as supporting information on the PNAS web site). A interacts with the first three strands of the next repeat unit to form canonical WD40 unit comprises a set of four antiparallel ␤-strands a single blade, and the propeller is closed by a blade formed by with a WD-like dipeptide at its C terminus, although identified units analogous interaction between the last strand of the last unit and the often vary greatly in sequence and the loops that connect consec- first three strands of the first unit (e.g., for Ski8, Fig. 4C)(20).By utive strands can vary greatly in length. The repeat units identified extension, Zip2 comprises 14 WD40-like repeats and thus could be in Zip2s all comprise four ␤-strands (by averaged consensus) and structurally analogous to Unc78͞Aip1, whose 14 WD40 repeats some, but not all, contain the diagnostic C-terminal dipeptide. form a linked pair of 7-blade propellers (21). These units are aligned to one another in Fig. 4A. Interestingly, whereas the metaserver gives no significant matches with full-length ABRA Defines APC Subunits Cdc20 and Cdh1 as 7-Blade WD40 Repeat Zip2s, the central region containing the initially observed aliphatic- Proteins. Cdc20 and Cdh1 are APC components and have previ- K-hydrophilic-L-X-W-D͞N motif is matched to a murine all-␤ Ig ously been identified as containing WD40 repeats. However, the structure, PDB ID code 1A7Q (18). precise numbers and limits of the repeat units have not been To further confirm the identification of 14 repeat units, we defined, and refined alignments among proteins in the two corre- subjected the corresponding alignment to phylogenetic analysis by sponding families have not been made. ABRA analysis reveals that TreeTop (www.genebee.msu.su͞services͞phtree࿝reduced.html) the WD40 domains of Cdc20 and Cdh1 are each composed of 7 (Fig. 4B). In many cases, consecutive repeats can be related to one units (Fig. 4A and Fig. 8, which is published as supporting infor- another at the terminal branches of a simple phylogenetic tree, mation on the PNAS web site). A publicly available structure-based

Perry et al. PNAS ͉ December 6, 2005 ͉ vol. 102 ͉ no. 49 ͉ 17597 Downloaded by guest on September 24, 2021 ABRA Defines 15-Unit TPR Arrays in APC Components Cdc16, Cdc23, and Cdc27. For further comparison, we applied ABRA to three of the relevant APC components whose TPRs, although presumptively present, remained unspecified: Cdc16, Cdc23, and Cdc27. Only sequences from the four organisms of the Zip2͞3͞Spo22͞ Zip4 cohort were analyzed. Cdc16s, Cdc23s, and Cdc27s all emerge as 15-unit TPR proteins (Figs. 10 and 11, which are published as supporting information on the PNAS web site). These TPR arrays are expected to be similar in general conformation to structurally analyzed arrays, e.g., the six-unit array from the vesicular transport protein Sec17 (Fig. 5B) (22).

Discussion Identification of an Additional ZMM Protein. Zip2 and Zip3 are formally defined as being part of a single functional unit, the ZMM group (2). Further, the two proteins interact physically and, at later stages of recombination, colocalize in prominent foci that mark the sites of developing crossovers (5, 6). We show here that spo22͞zip4 mutants confer the same diagnostic phenotypes as zip2, zip3, and other zmm mutants. These results, as well as cytological studies (G. S. Roeder, personal communication), define Zip4 as an additional member of the ZMM group.

An Evolutionarily Related Cohort of Zip2, Zip3, and Spo22͞Zip4 Pro- teins. Proteins having strong amino acid sequence homology to Zip2, Zip3, or Spo22͞Zip4 of budding yeast S. cerevisiae occur only in a small cohort of closely related organisms: K. lactis, C. glabrata, and C. albicans. ␣ Fig. 5. Application of ABRA to -repeat proteins. (A) Results of ABRA analysis It remains possible that divergent homologs of these proteins of Spo22 from S. cerevisiae. Spo22 emerges as a TPR protein. (B) Structural PSI BLAST example of a six-unit TPR polypeptide (Sec17, PDB ID code 1QQE). Rainbow exist in other organisms. For Zip3, a - search using the first colors roughly delimit successive TPR units. half of the sequence as a query yielded a number of below-threshold hits, e.g., in Drosophila melanogaster (AAF48955͞AAN14131͞ AAF45708), Drosophila pseudoobscura (EAL27394͞EAL32410), alignment (www.ncbi.nlm.nih.gov͞Structure͞cdd͞cddsrv.cgi? Caenorhabditis elegans (CAB00037), Neurospora crassa uidϭcd00200) reveals a subset of similar WD40 repeat units (EAA26717), and Gibberella zeae (EAA74630). All of these pro- identified by ABRA in Zip2, Cdc20, and Cdh1 (Fig. 4A). Both teins have RING fingers at their N termini and are predicted by approaches identify repeating structural elements of approximately PSORT (www.psort.org͞) to be nuclear (23). They are significantly the same length and sequence but with substantial tolerance for variable in size (Ϸ140 to Ͼ1,100 aa), but the differences come sequence degeneracy and high sequence variability of interunit primarily from low-complexity elements, also detectable in bona loops. WD40 domains of Cdc20 and Cdh1 thus emerge as pre- fide Zip3s, which may represent variable-sized linkers between sumptive 7-blade propellers structurally analogous to Ski8 (above). major globular domains. Further, the putative Caenorhabditis elegans gene was previously identified as a Zip3 candidate and ͞ ABRA Reveals Spo22 Zip4s as TPR Proteins. Despite the obvious phenotypic analysis suggests that this gene is required for comple- ͞ relationships among the four most homologous Spo22 Zip4 tion of recombination, analogous to yeast Zip3 (24). For Zip2 and sequences (above), an initial T-COFFEE alignment showed only Spo22͞Zip4, reasonable candidates for more-distantly related ho- 4.6% identity among them. Such disparities are frequently a mologs are not suggested by protein BLAST search algorithms with hallmark of the presence of a reiterated sequence motif. Cor- the exception of potential Spo22͞Zip4 candidates in N. crassa and respondingly, RADAR analysis of Spo22͞Zip4s indicates the Ϫ Cryptococcus neoformans (CAD37031, E ϭ 1e 04 and EAL19170, presence of repetitive elements. Further, PSI-BLAST finds statis- E ϭ 3eϪ04, respectively). Identification of distantly related Zip2 and tically significant homology to a number of TPR-containing Zip4 homologs is particularly difficult due to absence of any proteins (E Յ 0.001) after only the second iteration. Given these clues, we analyzed these sequences by ABRA. ABRA identifies diagnostic position-dependent catalytic modules and the presence 22 helix–loop–helix repetitive elements in the Spo22͞Zip4s of S. of structural repeats that share little sequence identity (3–5%) even cerevisiae and C. glabrata, 21 such elements in C. albicans and 18 among close homologs. such elements in K. lactis (Fig. 5A and Fig. 9, which is published as supporting information on the PNAS web site). The latter two Zip3 Has Motifs Found in Ubiquitin RING E3s and Is Homologous to sequences differ from the first two by apparent deletions at their Rad18. Zip3 is identified here as a RING E3 whose most probable N termini and thus might reflect gene annotation͞sequencing substrate is ubiquitin. Further, Zip3 emerges as homologous to errors rather than true sequence differences. Rad18, a known DNA-binding E3, which works in combination Three distinct types of helical repeat motifs have been described: with Rad6, a ubiquitin E2. Zip3 might exert its effects by means of HEAT, ARM, and TPR. ARM repeats are three-helix motifs and Rad6 and͞or some other partner. Interestingly, ubiquitin-mediated HEAT and TPR repeats are two-helix motifs. TPRs are distin- and SUMO-mediated protein modifications are often intimately guishable from HEAT repeats by the absence of a specific defining functionally linked (e.g., ref. 15). Moreover, we also find that S. bend near the middle of the N-terminal helix. Because only one of cerevisiae Zip3 contains a predicted high probability SUMOylation the repeat elements identified among the analyzed Spo22͞Zip4s is target consensus (Fig. 2A). Thus, complex functional relationships predicted to contain such a bend, these elements are most closely among Zip3, ubiquitination, and SUMOylation can be anticipated related to TPRs (as PSI-BLAST had suggested). (T.-F. Wang, personal communication).

17598 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508581102 Perry et al. Downloaded by guest on September 24, 2021 ABRA: A General Approach to Identification and Assignment of of this is the APC-mediated destruction of mitotic cyclins. However, Repeated Motifs in Biological Sequences. With ABRA, a standard more recently it has become apparent that both mono- and alignment is optimized manually by taking into account sequence polyubiquitinylation can instead serve as reversible signaling de- identities, amino acid properties and position, and predicted sec- vices, functionally analogous to phosphorylation. In some instances, ondary structure. Consecutive elements are marked only on the ubiquitin and SUMO compete for the same lysine residue on a basis of their repetitive nature, without respect to any defined given protein and are functionally antagonistic to one another. This sequence profile. This approach is particularly powerful if used in is an increasingly observed mechanism in chromosome-based ac- combination with an algorithm such as RADAR that can nucleate tivities, including transcription and DNA repair. For example, an array by locating cryptic repetitive elements in different locations monoubiquitinylation of a certain lysine residue on PCNA (prolif- in individual sequences; additional repeat segments can then be erating cell nuclear antigen) by Rad18, followed by Rad5-mediated inferred in all sequences at the corresponding positions of the ubiquitin chain extension, results in promoting polymerase activity alignment (e.g., as for Zip2s, above). Overall, this method produces to conduct error-free DNA repair. SUMOylation of the same an alignment-based structural model, where divergent sequences residue inhibits this process and results in the recruitment of the coalesce to yield a single averaged structure prediction. ABRA Srs2 helicase that disrupts Rad51 nucleoprotein filaments, presum- therefore differs significantly from the more commonly known ably to block recombination during S phase (15, 27). If the ZIP structure-based alignment methods in which a known structure is complex acts in protein degradation, then the roles of Zip2 and imposed on all available sequences. Spo22͞Zip4 are likely analogous to those of the WD40 and TPR proteins of the APC (Cdc20͞Cdh1 and Cdc16͞Cdc23͞Cdc27), Do Zip2, Zip3, and Spo22͞Zip4 Comprise a Ubiquitin Ligation Machine? namely, to serve as shuttle components to bring the substrate(s) Zip3 appears to be a ubiquitin E3 (above). Additional observations molecules into proximity of the Zip3 E3. However, if the ZIP suggest that Zip2 and Zip4 may also be involved in ubiquitin ligation complex acts to enhance or repress the activity of nearby molecules and, if so, in a somewhat unique way. by ubiquitin signaling, the roles of Zip2 and Spo22͞Zip4 are less Two of the major types of ubiquitin ligation complexes are the clear, and they may serve simply to help localize Zip3 to recom- SCF and the APC. Both contain a cullin scaffolding subunit, Cdc53 bination complexes. and Apc2, respectively. A search of publicly available S. cerevisiae two-hybrid screen databases (www.yeastgenome.org) reveals that Zip2͞3͞4 as a Locally Acting Ensemble. Most straightforwardly, Zip2 is a Cdc53-interacting protein. When Cdc53 is presented as effects of Zip2͞3͞4 on crossing-over and chiasma formation at the ‘‘bait’’ in these screens, there are more hits found with Zip2 (five) leptotene͞zygotene transition would arise by targeted ubiquitina- BIOCHEMISTRY than with any other protein, including anticipated hits such as the tion of chromatin, recombinosome components and͞or axial pro- F-box adaptor protein Skp1 (four); a protein implicated in cullin teins locally at the sites of progressing recombinational interactions. neddylation [Dcn1 (four)]; the F-box proteins Cdc4 (one), Met-30 Importantly, also, the phenotypes thus far described for zmm (one), and Mdm30 (four); and the ubiquitin E2, Cdc34 (one) (25). mutants all reflect a failure to properly execute the leptotene͞ Zip2-Cdc53 interactions are also identified in two-hybrid screens zygotene transition. However, the cytologically observable ensem- when Zip2 is presented as bait. Although not definitive, these bles (immunostaining foci) of Zip2 and Zip3 proteins appear findings suggest that Zip2–Cdc53 is a bona fide interaction. By concomitantly with this transition and are then found all along the extension these findings further suggest that Zip2 and Zip3 may SC during pachytene. This pattern suggests that immunostaining interact, with Cdc53, in a ubiquitin ligation complex. foci arise as a consequence of the leptotene͞zygotene transition (2). No two-hybrid interactions were identified between Cdc53 and Further, and more importantly, the corresponding proteins may Zip3, even though ubiquitin E3 proteins often interact directly with ͞ have additional functions at later step(s) of chiasma formation, cullin. However, many protein protein interactions detected by presumptively again acting locally to mediate changes at the chro- biochemical methods escape detection in two-hybrid screens, in- matin͞recombinosome͞axis levels. cluding the biochemically well documented association between ͞ ͞ yeast Cdc53 and the ubiquitin E3 Roc1 Hrt1 Rbx1 (26). We thank Scott Keeney for his insight regarding this work, Jim Henle for Ligation of ubiquitin to a target molecule can have at least two his assistance in manuscript preparation, Ting-Fang Wang for sharing functional consequences. The most widely studied effect is that data before publication, and Dimitri Sigounas and Geoff Fudenberg for polyubiquitin chain formation can target labeled molecules for assistance with some of the experiments. This work was supported by degradation by the 20S proteosome. A commonly known example National Institutes of Health Grants GM25326 and GM44794 (to N.K.).

1. Kauppi, L., Jeffreys, A. J. & Keeney, S. (2004) Nat. Rev. Genet. 5, 413–424. 15. Prakash, S., Johnson, R. E. & Prakash L. (2005) Annu. Rev. Biochem. 74, 2. Bo¨rner, G. V., Kleckner, N. & Hunter, N. (2004) Cell 117, 29–45. 317–353. 3. Hunter, N. & Kleckner, N. (2001) Cell 106, 59–70. 16. Notredame, C., Higgins, D. G. & Heringa J. (2000) J. Mol. Biol. 302, 4. Zickler, D. & Kleckner, N. (1999) Annu. Rev. Genet. 33, 603–754. 205–217. 5. Agarwal, S. & Roeder, G.S. (2000) Cell 102, 245–255. 17. Bailly, V., Prakash, S. & Prakash L. (1997) Mol. Cell Biol. 17, 4536–4543. 6. Henderson, K. A. & Keeney, S. (2004) Proc. Natl. Acad. Sci. USA 101, 4519–4524. 18. Ginalski, K., Elofsson, A., Fischer, D. & Rychlewski, L. (2003) Bioinformatics 7. Mazina, O. M., Mazin, A. V., Nakagawa, T., Kolodner, R. D. & Kowalc- 19, 1015–1018. zykowski, S. C. (2004) Cell 117, 47–56. 19. Heger, A. & Holm, L. (2000) Proteins 41, 224–237. 8. Ross-Macdonald, P., Coelho, P. S., Roemer, T., Agarwal, S., Kumar, A., 20. Madrona, A. Y. & Wilson, D. K. (2004) Protein Sci. 13, 1557–1565. Jansen, R., Cheung, K. H., Sheehan, A., Symoniatis, D., Umansky, L., et al. 21. Voegtli, W. C., Madrona, A. Y. & Wilson, D. K. (2003) J. Biol. Chem. 278, (1999) Nature 402, 413–418. 34373–34379. 9. McKee, A. H. & Kleckner, N. (1997) Genetics 146, 797–816. 22. Rice, L. M. & Brunger, A. T. (1999) Mol. Cell 4, 85–95. 10. Primig, M., Williams, R. M., Winzeler, E. A., Tevzadze, G. G., Conway, A. R., 23. Nakai, K. & Horton, P. (1999) Trends Biochem. Sci. 24, 34–36. Hwang, S. Y., Davis, R. W. & Esposito, R. E. (2000) Nat. Genet. 26, 415–423. 24. Jantsch, V., Pasierbek, P., Mueller, M. M., Schweizer, D., Jantsch, M. & Loidl, 11. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. J. (2004) Mol. Cell Biol. 24, 7998–8006. & Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402. 25. Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., 12. Sonnhammer, E. L., Eddy, S. R., Birney, E., Bateman, A. & Durbin, R. (1998) Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., et al. (2000) Nature 403, Nucleic Acids Res. 26, 320–322. 623–627. 13. Zhao, X. & Blo¨bel, G. (2005) Proc. Natl. Acad. Sci. USA 102, 4777–4782. 26. Ho, Y., Gruhler, A., Heilbut, A., Bader, G. D., Moore, L., Adams, S. L., Millar, 14. Miura, K., Rus, A., Sharkhuu, A., Yokoi, S., Karthikeyan, A. S., Raghothama, A., Taylor, P., Bennett, K., Boutilier, K., et al. (2002) Nature 415, 180–183. K. G., Baek, D., Koo, Y. D., Jin, J. B., Bressan, R. A., et al. (2005) Proc. Natl. 27. Pfander, B., Moldovan, G.-L., Sacher, M., Hoege, C. & Jentsch S. (2005) Nature Acad. Sci. USA 102, 7760–7765. 436, 428–433.

Perry et al. PNAS ͉ December 6, 2005 ͉ vol. 102 ͉ no. 49 ͉ 17599 Downloaded by guest on September 24, 2021