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1.	Astromskas, Eimantas, et al. (författare) Ends-in vs. ends-out targeted insertion mutagenesis in Saccharomyces castellii. 2009 Ingår i: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 55:3, s. 339-347 Tidskriftsartikel (refereegranskat)abstract Gene replacement (knock-out) is a major tool for the analysis of gene function. However, the efficiency of correct targeting varies between species, and is dependent on the structure of the DNA construct. We analyzed the targeted insertion mutagenesis method in the budding yeast Saccharomyces castellii, phylogenetically positioned after the whole genome duplication event in the Saccharomyces lineage. We compared the targeting efficiency for target DNA constructs in the respective ends-in and ends-out form. For some of the constructs S. castellii showed a similar high degree of homologous recombination as S. cerevisiae. In agreement with S. cerevisiae, a higher targeting efficiency was seen for the diploid strain than for the haploid. Surprisingly, a higher degree of targeting efficiency was seen for ends-out constructs compared to ends-in constructs. This result may have been influenced by the difference in the length of the homologous target sequences used, although long homology regions of 300 bp-1 kb were used in all constructs. Remarkably, very short regions of cohesive heterologous sequences at the ends of the constructs highly stimulated random illegitimate integration, suggesting that the pathway of non-homologous end joining is highly active in S. castellii.
2.	Astromskas, Eimantas, et al. (författare) Tools and methods for genetic analysis of Saccharomyces castellii 2007 Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 24:6, s. 499-509 Tidskriftsartikel (refereegranskat)abstract The budding yeast species Saccharomyces castellii has provided important new insights into molecular evolution when incorporated in comparative genomics studies and studies of mitochondrial inheritage. Although it shows some diversity in the specific molecular details, several analyses have shown that it contains many genetic pathways similar to those of S. cerevisiae. Here we have investigated the possibility of performing genetic analyses in S. castellii. We optimized the LiAc transformation protocol to achieve 200-300 transformants/mu g plasmid DNA. We found that the commonly used plasmids for S. cerevisiae are stably maintained in S. castellii under selective conditions. Surprisingly, both 2 mu and CEN/ARS plasmids are kept at a high copy number. Moreover, the kanMX cassette can be used as a resistance marker against the selective drug geneticin (G418). Finally, we determined that the S. cerevisiae GAL] promoter can be used for the activation of transcription in S. castellii, thus enabling the controlled overexpression of genes when galactose is present in the medium. The availability of these tools provides the possibility or performing genetic analyses in S. castellii, and makes it a promising new model system in which hypotheses derived from bioinformatics studies can be experimentally tested. Copyright (C) 2007 John Wiley & Sons, Ltd.
3.	Cohn, Marita, et al. (författare) Alternative Lengthening of Telomeres in the Budding Yeast Naumovozyma castellii 2019 Ingår i: G3: Genes, Genomes, Genetics. - : Oxford University Press (OUP). - 2160-1836. ; 9:10, s. 3345-3358 Tidskriftsartikel (refereegranskat)abstract The enzyme telomerase ensures the integrity of linear chromosomes by maintaining telomere length. As a hallmark of cancer, cell immortalization and unlimited proliferation is gained by reactivation of telomerase. However, a signiﬁcant fraction of cancer cells instead uses alternative telomere lengthening mechanisms toensuretelomere function,collectively known asAlternative Lengthening ofTelomeres(ALT). Although the budding yeast Naumovozyma castellii (Saccharomyces castellii) has a proﬁcient telomerase activity, we demonstrate here that telomeres in N. castellii are efﬁciently maintained by a novel ALT mechanism after telomerase knockout. Remarkably, telomerase-negative cells proliferate indeﬁnitely without any major growth crisis and display wild-type colony morphology. Moreover, ALT cells maintain linear chromosomes and preserve a wild-type DNA organization at the chromosome termini, including a short stretch of terminal telomeric sequence. Notably, ALT telomeres are elongated by the addition of 275 bp repeats containing a short telomeric sequence and the subtelomeric DNA located just internally (TelKO element). Although telomeres may be elongated by several TelKO repeats, no dramatic genome-wide ampliﬁcation occurs, thus indicating that the repeat addition may be regulated. Intriguingly, a short interstitial telomericsequence(ITS)functionsastheinitiationpointfortheadditionoftheTelKOelement.This implies that N. castellii telomeres are structurally predisposed to efﬁciently switch to the ALT mechanism as a response to telomerase dysfunction.
4.	Cohn, Marita, et al. (författare) Chromosome ends in Chironomus pallidivittatus contain different subfamilies of telomere-associated repeats 1992 Ingår i: Chromosoma. - 0009-5915. ; 101:10, s. 634-640 Tidskriftsartikel (refereegranskat)abstract Tandemly repeated 340 bp sequences, TA repeats, are present in seven of the eight pairs of chromosome ends in Chironomus pallidivittatus, being absent from the telocentric left end of chromosome four. We have previously shown that the family of TA repeats consists of four main subfamilies. One subfamily is composed of a master unit and the other three contain derived units, each of which has a small region where the master sequence is highly mutated. Here we find that there are considerable variations in numbers of TA repeats between animals and for the same telomere in different animals. We also show that the seven telomere pairs containing TA repeats differ with regard to the content of derived subfamilies. The master unit is probably present in all seven pairs. Two of the derived units are exclusively present in two telomere pairs. The third derived unit shows a more irregular distribution. Some of the telomeres have highly variable contents of such units among animals. Subfamilies thus have different behaviour as reflected in their stable and variable patterns of distribution between individual telomeres.
5.	Cohn, Marita, et al. (författare) Evolutionary relations between subtypes of telomere-associated repeats in Chironomus 1991 Ingår i: Journal of Molecular Evolution. - 0022-2844. ; 32:6, s. 463-468 Tidskriftsartikel (refereegranskat)abstract Telomere-associated DNA in Chironomus pallidivittatus contains tandemly repeated 340-bp units. We show that they are distributed among several subtypes of which we have characterized two, M1 and D1, with regard to base sequence, homogeneity, and intertelomeric distribution. Each subpopulation is highly homogeneous and the two subtypes have identical consensus sequences throughout 90% of their lengths. In the remaining part the homology is only about 60%. Each subpopulation has its specific intertelomeric distribution and there is no difference in the degree of homogenization within and between telomeres. The repeat unit contains two pairs of subrepeats embedded in linker DNA. This provides a model that makes it possible to relate the two subtypes to each other with regard to evolutionary history. The difference between the two subtypes is due to mutations that have occurred in only one of them, D1, resulting in a decreased similarity between one of its pairs of subrepeats. This type of repeat unit is therefore believed to be derived from the other, M1. The local decrease in similarity between M1 and D1 suggests that homogenization between them occurs by gene conversion.
6.	Cohn, Marita, et al. (författare) Molecular diversity of telomeric sequences 2008 Ingår i: Origin and evolution of telomeres. - 9781587063091 Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract Telomeric DNA generally comprises a repetitive, noncoding sequence which terminates in a single‑stranded extension, the 3' overhang. Although these species‑specific repeats show a large diversity in sequence and length, they comprise some specific characteristic conserved features. Therefore, analyses of the telomeric repeats of various organisms have delivered important insights concerning the origin and evolution of telomere structures. The canonical repeats are synthesized onto the 3' overhangs by the specialized telomerase enzyme. Although the general feature of the telomerase is to produce homogeneous repeats, variant repeats and irregular sequences are found in many species. In this chapter some aspects of the conserving and diverging evolutionary forces on the telomeric sequences are discussed. Proteins specifically binding to the telomeric sequences are of importance for nucleating the cap structure and they regulate telomere homeostasis and telomere end protection. Since a conserved binding specificity has been shown in several homologues of both single‑stranded and double‑stranded telomere‑binding proteins, their binding may be considered a major selective force operating on telomeric DNA sequences.
7.	Cohn, Marita (författare) OB Fold Contributes to Telomere Maintenance. 2013 Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 21:1, s. 3-4 Tidskriftsartikel (refereegranskat)abstract The essential Cdc13 protein is part of the trimeric CST complex that confers genome stability by binding to and protecting yeast telomeres. In this issue of Structure, Mason and colleagues characterize an OB fold domain of Cdc13 (named OB2) and propose that homo-dimerization of OB2 is required for proper assembly of the CST complex and telomere maintenance.
8.	Cohn, Marita, et al. (författare) Saccharomyces cerevisiae RAP1 bind to telomeric sequences with spatial flexibility 2000 Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962. ; 28:12, s. 2292-2301 Tidskriftsartikel (refereegranskat)abstract A wide divergence has been detected in the telomeric sequences among budding yeast species. Despite their length and homogeneity differences, all these yeast telomeric sequences show a conserved core which closely matches the consensus RAP1-binding sequence. We demonstrate that the RAP1 protein binds this sequence core, without involving the diverged sequences outside the core. in Saccharomyces castellii and Saccharomyces dairensis specific classes of interspersed variant repeats are present. We show here that a RAP1-binding site is formed in these species by connecting two consecutive 8 bp telomeric repeats. DNase I footprint analyses specify the binding site as the 13 bp sequence CTGGGT-GTCTGGG, The RAP1 protein also binds the variant repeats, although with a lowered affinity. However, a split footprint is produced when RAP1 binds a variant repeat where the two half-sites of the binding site are separated by an additional 6 nt. This is probably caused by the intervening sequence looping out of the RAP1-DNA complex. We suggest that the bipartite subdomain structure of the RAP1 protein allows it to remodel telomeric chromatin, a feature which may be of great relevance for telomeric chromatin assembly and structure in vivo.
9.	Cohn, Marita, et al. (författare) Telomere-associated repeats in Chironomus form discrete subfamilies generated by gene conversion 1992 Ingår i: Journal of Molecular Evolution. - 0022-2844. ; 35:2, s. 114-122 Tidskriftsartikel (refereegranskat)abstract In dipteran insects the most distal telomere-associated DNA known to exist consists of long, complex tandem repeats. We have classified the 340-bp tandemly arranged repeats in Chironomus pallidivittatus. The repeats are distributed in a small number of subfamilies. One type of the repeat has the character of a master unit from which other main units can be derived usually by simple changes. The derived subfamilies contain segments that are degenerate versions of the corresponding segment in the master sequence. Such segments can also occur together in one and the same repeat unit in different combinations. There is a complete absence of subfamily-specific base variants in regions lying outside of the degenerate segments. Homogenization takes place between DNA sequences that are often smaller than a whole repeat unit. The mosaic structure of the repeat arrays suggests that gene conversion is an important force in the generation and maintenance of this family of repeats.
10.	Cohn, Marita, et al. (författare) Telomeres in fungi 2006 Ingår i: Comparative genomics using fungi as models. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 1610-2096 .- 1610-6970. - 9783540314806 - 9783540314950 ; 15, s. 100-130 Bokkapitel (refereegranskat)
11.	Cohn, Marita, et al. (författare) Telomeric sequence diversity within the genus Saccharomyces 1998 Ingår i: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 33:2, s. 83-91 Tidskriftsartikel (refereegranskat)abstract Conservation of telomeric DNA repeat sequences has been found across evolutionarily diverse eukaryotes. Here we report on a marked telomeric sequence diversity within the budding yeast genus Saccharomyces. Cloning and sequencing of telomeric repeat units from S. castellii, S. dairensis, S. exiguus and S. kluyveri showed a length variation between 8 and 26 bp, as well as a distinct variation in the degree of homogeneity, among the species. In S. castellii and S. dairensis, TCTGGGTG constituted a majority of the telomeric repeat units. However, the character of the variant repeats differed: in S. castellii the major class of variant repeats contained additional TG dinucleotides per repeat unit, [TCTGGGTG(TG)(1-3)], whereas in S. dairensis the major variant repeat is the shorter, uniform sequence TCTGGG. This result suggests mechanistic differences in the action of the telomerases of these closely related yeasts. Despite their length and homogeneity differences, all the Saccharomyces telomeric sequences show a conserved core which is also shared by the Candida glabrata telomeric sequence. This evolutionary similarity may be partly explained by the preservation of a binding site for the RAP1 protein.
12.	Fridholm, Helena, et al. (författare) Telomerase-dependent generation of 70-nt-long telomeric single-stranded 3' overhangs in yeast 2012 Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. Tidskriftsartikel (refereegranskat)abstract Telomeres, the chromatin structures at the ends of eukaryotic chromosomes, are essential for chromosome stability. The telomere terminates with a TG-rich 3' overhang, which is bound by sequence-specific proteins that both protect the end and regulate the telomerase elongation process. Here, we demonstrate the presence of 3' overhangs as long as 200 nt in asynchronously growing cells of the budding yeast Saccharomyces castellii. The 3' overhangs show a wide distribution of 14-200 nt in length, thus resembling the distribution found in human cells. A substantially large fraction of the 3' overhangs resides in the 70-200 nt range. Remarkably, we found an accumulation of a distinct class of 70-nt-long 3' overhangs in the S phase of the cell cycle. Cells without a functional telomerase showed the same wide distribution of 3' overhangs, but significantly, lacked the specific fraction of 70-nt 3' overhangs. Hence, our data show that the highly defined 70-nt 3' overhangs are generated by a telomerase-dependent mechanism, which is uncoupled to the mechanisms producing the bulk of the 3' overhangs. These data provide new insights that will be helpful for deciphering the complex interplay between the specialized telomere replication machinery and the conventional DNA replication.
13.	Gustafsson, Cecilia, et al. (författare) Rap1 Binds Single-stranded DNA at Telomeric Double- and Single-stranded Junctions and Competes with Cdc13 Protein 2011 Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 286:52, s. 45174-45185 Tidskriftsartikel (refereegranskat)abstract The ends of eukaryotic chromosomes are protected by specialized telomere chromatin structures. Rap1 and Cdc13 are essential for the formation of functional telomere chromatin in budding yeast by binding to the double-stranded part and the single-stranded 3' overhang, respectively. We analyzed the binding properties of Saccharomyces castellii Rap1 and Cdc13 to partially single-stranded oligonucleotides, mimicking the junction of the double- and single-stranded DNA (ds-ss junction) at telomeres. We determined the optimal and the minimal DNA setup for a simultaneous binding of Rap1 and Cdc13 at the ds-ss junction. Remarkably, Rap1 is able to bind to a partially single-stranded binding site spanning the ds-ss junction. The binding over the ds-ss junction is anchored in a single double-stranded hemi-site and is stabilized by a sequence-independent interaction of Rap1 with the single-stranded 3' overhang. Thus, Rap1 is able to switch between a sequence-specific and a nonspecific binding mode of one hemi-site. At a ds-ss junction configuration where the two binding sites partially overlap, Rap1 and Cdc13 are competing for the binding. These results shed light on the end protection mechanisms and suggest that Rap1 and Cdc13 act together to ensure the protection of both the 3' and the 5' DNA ends at telomeres.
14.	Itriago, Humberto, et al. (författare) Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii 2023 Ingår i: Genes. - 2073-4425. ; 14:10 Tidskriftsartikel (refereegranskat)abstract Several sources of DNA damage compromise the integrity and stability of the genome of every organism. Specifically, DNA double-strand breaks (DSBs) can have lethal consequences for the cell. To repair this type of DNA damage, the cells employ homology-directed repair pathways or non-homologous end joining. Homology-directed repair requires the activity of the RAD52 epistasis group of genes. Rad52 is the main recombination protein in the budding yeast Saccharomyces cerevisiae, and rad52Δ mutants have been characterized to show severe defects in DSB repair and other recombination events. Here, we identified the RAD52 gene in the budding yeast Naumovozyma castellii. Our analysis showed that the primary amino acid sequence of N. castellii Rad52 shared 70% similarity with S. cerevisiae Rad52. To characterize the gene function, we developed rad52Δ mutant strains by targeted gene replacement transformation. We found that N. castellii rad52Δ mutants showed lowered growth capacity, a moderately altered cell morphology and increased sensitivity to genotoxic agents. The decreased viability of the N. castellii rad52Δ mutants in the presence of genotoxic agents indicates that the role of the Rad52 protein in the repair of DNA damage is conserved in this species.
15.	Karademir Andersson, Ahu, et al. (författare) Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii) 2016 Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 33:12, s. 633-646 Tidskriftsartikel (refereegranskat)abstract The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies, functional analyses of centromere DNA elements, and was shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type (MATa or MATα) was determined for each hoΔ mutant, and was stable under sporulation inducing conditions, showing that the switching system was totally non-functional. The hoΔ strains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔ strains were mated, resulting in diploid cells that efficiently formed asci and generated viable spores when microdissected. By introduction of a point mutation in the URA3 gene, we created a uracil auxotrophic strain, and by exchanging the kanMX3 cassette for the hphMX4 cassette we show that hygromycin B resistance can be used as a selection marker in N. castellii. These haploid strains containing genetic markers will be useful tools for doing genetic analyses in N. castellii. Moreover, we demonstrate that homology regions of 200-230 bp can be successfully used for target site-specific integration into genomic loci.
16.	Karademir Andersson, Ahu, et al. (författare) Multiple DNA Interactions Contribute to the Initiation of Telomerase Elongation 2017 Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 429:14, s. 2109-2123 Tidskriftsartikel (refereegranskat)abstract Telomerase maintains telomere length and chromosome integrity by adding short tandem repeats of single-stranded DNA to the 3' ends, via reverse transcription of a defined template region of its RNA subunit. To further understand the telomerase elongation mechanism, we studied the primer utilization and extension activity of the telomerase from the budding yeast Naumovozyma castellii (Saccharomyces castellii), which displays a processive nucleotide and repeat addition polymerization. For the efficient initiation of canonical elongation, telomerase required 4-nt primer 3' end complementarity to the template RNA. This DNA-RNA hybrid formation was highly important for the stabilization of an initiation-competent telomerase-DNA complex. Anchor site interactions with the DNA provided additional stabilization to the complex. Our studies indicate three additional separate interactions along the length of the DNA primer, each providing different and distinct contributions to the initiation event. A sequence-independent anchor site interaction acts immediately adjacent to the base-pairing 3' end, indicating a protein anchor site positioned very close to the catalytic site. Two additional anchor regions further 5' on the DNA provide sequence-specific contributions to the initiation of elongation. Remarkably, a non-telomeric sequence in the distal 25- to 32-nt region negatively influences the initiation of telomerase elongation, suggesting an anchor site with a regulatory role in the telomerase elongation decision.
17.	Karademir Andersson, Ahu, et al. (författare) Naumovozyma castellii: an alternative model for budding yeast molecular biology 2017 Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 34:3, s. 95-109 Tidskriftsartikel (refereegranskat)abstract Naumovozyma castellii (Saccharomyces castellii) is a member of the budding yeast family Saccharomycetaceae. It has been extensively used as a model organism for telomere biology research and has gained an increasing interest as a budding yeast model for functional analyses owing to its amenability to genetic modifications. Due to the suitable phylogenetic distance to S. cerevisiae the whole genome sequence of N. castellii has provided unique data for comparative genomic studies, and it played a key role in the establishment of the timing of the whole genome duplication and the evolutionary events that took place in the subsequent genomic evolution of the Saccharomyces lineage. Here we summarize the historical background of its establishment as a laboratory yeast species, and the development of genetic and molecular tools and strains. We review the research performed on N. castellii, focusing on areas where it has significantly contributed to the discovery of new features of molecular biology and to the advancement of our understanding of molecular evolution.
18.	Passoth, Volkmar, et al. (författare) Analysis of the hypoxia-induced ADH2-promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast 2003 Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 20:1, s. 39-51 Tidskriftsartikel (refereegranskat)abstract We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP. This system was used to analyse hypoxia-dependent PsADH2 regulation. Reporter gene activity was only found under oxygen limitation on a fermentable carbon source. The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae. Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction. This motif was different from the known hypoxia response elements of S. cerevisiae but showed some similarity to the mammalian HIF-1 binding site. Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation. Similar to the S. cerevisiae heme sensor system, the promoter was induced by Co2+. Cyanide was not able to mimic the effect of oxygen limitation. The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co2+ but not by cyanide. Thus, the very first promoter analysis in P. stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast.
19.	Rhodin, Jenny, et al. (författare) Characterization of the DNA binding features of Saccharomyces castellii Cdc13p 2006 Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 355:3, s. 335-346 Tidskriftsartikel (refereegranskat)abstract The principal function of Saccharomyces cerevisiae Cdc13p is to provide a loading platform to recruit complexes that provide end protection and telomere replication. We isolated the Saccharomyces castellii Cdc13p homolog (scasCdc13p) and characterized the in vitro DNA binding features of the purified recombinant scasCdc13p. The full-length scasCdc13p binds specifically to G-rich single-stranded telomeric DNA, and not to double-stranded DNA or the C-rich strand. Moreover, the minimal binding site for scasCdc13p is the octamer 5'-GTGTCTGG-3' of the S. castellii telomeric sequence. The scasCdc13p displayed a high affinity binding, where four individual nucleotide residues were found to be of most importance for the sequence specificity. Nonetheless, scasCdc13p binds the telomeric repeats from various other species, including the human. In spite of considerable divergence in telomere repeat length and sequence between these species, a conserved Cdc13p binding motif was detected. Among the budding yeasts this conserved Cdc13p binding site overlaps the Rap1p binding site. Together, these data implicate scasCdc13p as a telomere end-binding protein with a potential role in the regulation of telomere maintenance in vivo. Moreover, the results suggest that Rap 1p and Cdc13p act together to preserve the conserved core present within the otherwise highly divergent btelomeric sequences among a wide variety of yeasts.
20.	Rhodin, Jenny, et al. (författare) Highly sequence-specific binding is retained within the DNA-binding domain of the Saccharomyces castellii Cdc13 telomere-binding protein 2008 Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 8:8, s. 1289-1302 Tidskriftsartikel (refereegranskat)abstract The essential protein Cdc13p binds the single-stranded telomeric 3' overhangs in Saccharomyces cerevisiae and takes part in the regulation of telomere length. The DNA-binding domain (DBD) of Cdc13p is structurally established by an oligonucleotide/oligosaccharide-binding (OB)-fold domain. The sequence homolog in Saccharomyces castellii (scasCDC13) was characterized previously, and the full-length protein was found to bind telomeric DNA specifically. Here, the DBD of scasCdc13p was defined to the central part (402-658) of the protein. The region necessary for forming the scasCdc13p-DBD is larger than the minimal DBD of S. cerevisiae Cdc13p. Deletion of this extended DBD region from the full-length protein completely abolished the DNA binding, indicating the importance of the extended region for the correct formation of a binding-competent DBD. The scasCdc13p-DBD bound the same 8-mer minimal binding site as the full-length protein, but an extension of the target site in the 3' end increased the stability of the DNA-protein complex. Significantly, scasCdc13p-DBD showed a retained high sequence specific binding, where the four nucleotides of most importance for the sequence specificity are highly conserved in eukaryotic telomeric repeats. Thus, the unique single-stranded DNA-binding properties of the full-length protein are entirely retained within the isolated scasCdc13p-DBD.
21.	Rhodin, Jenny, et al. (författare) Single- and double-stranded DNA binding proteins act in concert to conserve a telomeric DNA core sequence 2011 Ingår i: Genome Integrity. - : Springer Science and Business Media LLC. - 2041-9414. ; 2:1 Tidskriftsartikel (refereegranskat)abstract Telomeres are protective cap structures at the ends of the linear eukaryotic chromosomes, which provide stability to the genome by shielding from degradation and chromosome fusions. The cap consists of telomere-specific proteins binding to the respective single- and double-stranded parts of the telomeric sequence. In addition to the nucleation of the chromatin structure the telomere-binding proteins are involved in the regulation of the telomere length. However, the telomeric sequences are highly diverged among yeast species. During the evolution this high rate of divergency presents a challenge for the sequence recognition of the telomere-binding proteins.ResultsWe found that the Saccharomyces castellii protein Rap1, a negative regulator of telomere length, binds a 12-mer minimal binding site (MBS) within the double-stranded telomeric DNA. The sequence specificity is dependent on the interaction with two 5 nucleotide motifs, having a 6 nucleotide centre-to-centre spacing. The isolated DNA-binding domain binds the same MBS and retains the same motif binding characteristics as the full-length Rap1 protein. However, it shows some deviations in the degree of sequence-specific dependence in some nucleotide positions. Intriguingly, the positions of most importance for the sequence-specific binding of the full-length Rap1 protein coincide with 3 of the 4 nucleotides utilized by the 3' overhang binding protein Cdc13. These nucleotides are very well conserved within the otherwise highly divergent telomeric sequences of yeasts.ConclusionsRap1 and Cdc13 are two very distinct types of DNA-binding proteins with highly separate functions. They interact with the double-stranded vs. the single-stranded telomeric DNA via significantly different types of DNA-binding domain structures. However, we show that they are dependent on coinciding nucleotide positions for their sequence-specific binding to telomeric sequences. Thus, we conclude that during the molecular evolution they act together to preserve a core sequence of the telomeric DNA.
22.	Rosas Bringas, Fernando Rodrigo, et al. (författare) Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in Saccharomyces cerevisiae 2022 Ingår i: eLife. - 2050-084X. ; 11 Tidskriftsartikel (refereegranskat)abstract Rap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. Rif2 and the Ku complex work in parallel to prevent tlc1-tm telo-mere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.
23.	Runnberg, Rikard, et al. (författare) Either Rap1 or Cdc13 can protect telomeric single-stranded 3' overhangs from degradation in vitro. 2019 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9 Tidskriftsartikel (refereegranskat)abstract Telomeres, the DNA-protein structures capping the ends of linear chromosomes, are important for regulating replicative senescence and maintaining genome stability. Telomeres consist of G-rich repetitive sequences that end in a G-rich single-stranded (ss) 3′ overhang, which is vital for telomere function. It is largely unknown how the 3′ overhang is protected against exonucleases. In budding yeast, double-stranded (ds) telomeric DNA is bound by Rap1, while ssDNA is bound by Cdc13. Here, we developed an in vitro DNA 3′end protection assay to gain mechanistic insight into how Naumovozyma castellii Cdc13 and Rap1 may protect against 3′ exonucleolytic degradation by Exonuclease T. Our results show that Cdc13 protects the 3′ overhang at least 5 nucleotides (nt) beyond its binding site, when bound directly adjacent to the ds-ss junction. Rap1 protects 1–2 nt of the 3′ overhang when bound to dsDNA adjacent to the ds-ss junction. Remarkably, when Rap1 is bound across the ds-ss junction, the protection of the 3′ overhang is extended to 6 nt. This shows that binding by either Cdc13 or Rap1 can protect telomeric overhangs from 3′ exonucleolytic degradation, and suggests a new important role for Rap1 in protecting short overhangs under circumstances when Cdc13 cannot bind the telomere.
24.	Runnberg, Rikard, et al. (författare) Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends 2017 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1 Tidskriftsartikel (refereegranskat)abstract Telomere DNA ends with a single-stranded 3′ overhang. Long 3′ overhangs may cause aberrant DNA damage responses and accelerate telomere attrition, which is associated with cancer and aging, respectively. Genetic studies have indicated several important players in preventing 5′ end hyper-resection, yet detailed knowledge about the molecular mechanism in which they act is still lacking. Here, we use an in vitro DNA 5′ end protection assay, to study how N. castellii Cdc13 and Rap1 protect against 5′ exonucleolytic degradation by λ-exonuclease. The homogeneous telomeric repeat sequence of N. castellii allows us to study their protection ability at exact binding sites relative to the 5′ end. We find efficient protection by both Cdc13 and Rap1 when bound close to the 5′ end. Notably, Rap1 provides protection when binding dsDNA at a distance from the 5′ end. The DNA binding domain of Rap1 is sufficient for 5′ end protection, and its wrapping loop region is essential. Intriguingly, Rap1 facilitates protection also when its binding site contains 2 nt of ssDNA, thus spanning across the ds-ss junction. These results highlight a role of Rap1 in 5′ end protection and indicate that Cdc13 and Rap1 have complementary roles in maintaining proper 3′ overhang length.
25.	Wahlin, Johan, et al. (författare) Analysis of the RAP1 protein binding to homogeneous telomeric repeats in Saccharomyces castellii. 2002 Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 19:3, s. 241-256 Tidskriftsartikel (refereegranskat)abstract The repressor activator protein 1 (RAP1) plays a role in telomere structure and function inS. cerevisiae. Here, the RAP1 homologue was identified and cloned from the budding yeast Saccharomyces castellii (scasRAP1). The scasRAP1 gene encodes a protein of 826 amino acids and shares an overall high degree of similarity with the S. cerevisiae RAP1 (scerRAP1). We demonstrate that the scasRAP1 is able to complement scerRAP1 in temperature-sensitive S. cerevisiae strains and is able to function as a regulator to maintain the original telomere lengths. Binding analyses of the E. coli-expressed scasRAP1 protein demonstrate that it needs two consecutive telomeric repeats in order to bind the S. castellii telomeric DNA sequences, and that it binds adjacent sites having a 16 bp centre-to-centre spacing. The binding affinity to telomeric DNA of several other yeasts is similar to that of scerRap1p. However, in contrast to scerRap1p, scasRap1p was found to bind the human telomeric sequence. Moreover, the scasRap1p was found to incorporate a variant repeat in its binding to the otherwise homogeneous telomeric DNA of S. castellii. This ability to bind various sites differing in DNA sequence indicates a high degree of adjustability in the binding of scasRap1p to DNA.
26.	Wahlin, Johan, et al. (författare) DNA binding and telomere length regulation of yeast RAP1 homologues. 2003 Ingår i: Journal of Molecular Biology. - 1089-8638. ; 332:4, s. 821-833 Tidskriftsartikel (refereegranskat)
27.	Wahlin, Johan, et al. (författare) RAP1 binding and length regulation of yeast telomeres 2002 Ingår i: Telomerases, telomeres and Cancer. - 0306474379 ; 22, s. 259-281 Bokkapitel (övrigt vetenskapligt/konstnärligt)
28.	Zhang, Y-J, et al. (författare) A family of complex tandem DNA repeats in the telomeres of Chironomus pallidivittatus 1994 Ingår i: Molecular and Cellular Biology. - 0270-7306. ; 14:12, s. 8028-8036 Tidskriftsartikel (refereegranskat)abstract A family of 340-bp tandem telomere-associated DNA repeats is present in 50- to 200-kb blocks in seven of the eight paired chromosome ends in Chironomus pallidivittatus. It consists of four main subfamilies, differing from each other by small clusters of mutations. This differentiation may reflect different functional roles for the repeats. Here we find that one subfamily, D3, is consistently localized most peripherally and extends close to the ends of the chromosomes, as shown by its sensitivity to the exonuclease Bal 31. The amounts of D3 are highly variable between individuals. The repeat characteristic for D3 forms a segment with pronounced dyad symmetry, which in single-strand form would give rise to a hairpin. Evidence from an interspecies comparison suggests that a similar structure is the result of selective forces. Another subfamily, M1, is present more proximally in a subgroup of telomeres characterized by a special kind of repeat variability. Thus, a complex block with three kinds of subfamilies may occupy different M1 telomeres depending on the stock of animals. We conclude that subfamilies are differentially distributed between and within telomeres and are likely to serve different functions.

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