Fungal Genetics and Biology 45 (2008) S54–S62

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Fungal Genetics and Biology

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Review

Predicted elements of telomere organization and function in Ustilago maydis

Patricia Sánchez-Alonso b, Plinio Guzman a,*
a
Departamento de Ingeniería Genética de Plantas, Cinvestav Campus Guanajuato, Apdo. Postal 629, Irapuato, Gto. 36821, Mexico
b
Centro de Investigaciones Microbiológicas, Instituto de Ciencias, BUAP Av. San Claudio y 18 sur, Edif. 76 3er piso, C.U. Puebla, Pue. 72570, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Telomeres are specialized caps of nucleoprotein complexes located at the chromosome termini. They
Received 4 December 2007 consist of short DNA repeats and of an assortment of associated proteins whose function is currently
Accepted 11 April 2008 under intense investigation in model systems. These specialized structures protect the linear ends of
Available online 29 May 2008
eukaryotic chromosomes against DNA repair and degradation activities, and serve as the substrate for tel-
omerase, the ribonucleoprotein complex that synthesises the telomere repeats. The pivotal role of the
Keywords: telomeres in the maintenance of cell viability in several model eukaryotes, including humans, greatly
Basidiomycete
promoted research in telomere biology. Studies on telomere structure and function in fungi other than
Fungi
Chromosome organization
model systems are limited to providing information on the telomeric repeat sequences. Here, we have
Telomere-associated sequences summarized the current knowledge on the organization of chromosome ends and on the proteins partic-
Telomerase ipating in telomere function in model systems including recent information obtained for filamentous
RecQ helicase fungi. We also describe Ustilago maydis genes that are potential homologs of proteins known from other
Contingency genes systems to participate in telomere biology.
Telosome Ó 2008 Elsevier Inc. All rights reserved.
Shelterin
Epigenetic silencing
DNA-damage response

1. Introduction sequence. A less common mechanism to maintain telomeres is

Every eukaryotic chromosome is assembled from a DNA mole-
cule associated with proteins that compact the DNA to be stored
in the nucleus of the cell. An array of tens or hundreds of copies
of a distinct simple DNA sequence is tandemly repeated at the
two ends of the DNA molecule, this assembly is known as the telo-
mere. In most organisms, except in Drosophila where chromo-
some-ends specific retrotransposons were recruited to carry out
telomere functions, the synthesis, composition and functioning of
telomeres share various conserved features among all eukaryotic
organisms (Gilson and Geli, 2007; Villasante et al., 2007).
Current knowledge of telomere biology comes from the pioneer-
ing work on protozoans and Saccharomyces cerevisiae and subse-
quently on mammalian cells (Greider, 1998). In the late seventies,
Elizabeth H. Blackburn discovered that Tetrahymena telomeres con-
sisted of a short DNA sequence motif that was repeated several times
at the chromosomal end (Blackburn and Gall, 1978). This pattern
was then found conserved throughout eukaryotes. The telomerase,
a polymerase involved in the synthesis of the telomere repeats was
identified a few years later (Lingner et al., 1997). Telomerase is a
specialized reverse transcriptase that includes a RNA component
required as the template for synthesis of the telomeric repeated
* Corresponding author. Fax: +52 462 6245849.
E-mail address: pguzman@ira.cinvestav.mx (P. Guzman).
known as ALT (alternative lengthening of telomeres), and involves
homologous recombination between telomeric repeats in order to
extend the chromosomal ends (Reddel, 2003).
The discovery of telomerase ignited research on telomere biol-
ogy (Blackburn et al., 2006). Soon it was evident a critical role that
these protective caps at chromosome-ends had in the maintenance
of cell viability in various model eukaryotes and it was clear that
the regulation of telomere length is a complex task for the cell.
Most cells’ telomeres shorten in every round of replication. Since
very short telomeres result in cell cycle arrest, telomere length is
thought to be a rate-limiting factor for lifespan and cell survival
(Bodnar et al., 1998; Vaziri and Benchimol, 1998).
The telomerase is a highly regulated enzyme whose expression
and activity are strictly regulated in the metazoans as well as in
unicellular eukaryotes (Cech, 2004; Greider and Blackburn,
2004). The capability of cells to replicate has been associated with
the functioning of this enzyme and with the extension of the telo-
meres. Telomerase activity is practically undetectable in the major-
ity of human somatic cells, while most carcinogenic tumor cells,
germ cells, and stem cells show significant telomerase activity
(Kim et al., 1994; Wright et al., 1996). Thus, this sort of evidence
correlates telomerase activity with malignant cellular transforma-
tion (Sharpless and DePinho, 2007). Conversely, the absence of tel-
omerase activity in mesenchymal stem cells of mouse hinders
cellular differentiation (Liu et al., 2004).

1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2008.04.009
 P. Sánchez-Alonso, P. Guzman / Fungal Genetics and Biology 45 (2008) S54–S62 S55

Telomere-associated proteins, known as the telosome in S. cere- Table 1

visiae and shelterin in humans, initially thought only to be involved Telomere repeat motifs in fungi

in telomere protection and telomerase access, have pivotal roles in Organism Telomere repeat Reference
regulating telomere length and replication (Karlseder, 2006). For Aspergillus nidulans TTAGGG a
instance, the shelterin complex, protects the telomere and is also Pneumocystis carinii TTAGGG b
required for telomere length control, recruiting and releasing telo- Metarrhizium anisopliae TTAGGG c
merase from telomere 30 overhangs (de Lange, 2005). POT1 and Magnaporthe grisea TTAGGG d
Neurospora crassa TTAGGG e
TPP1, two components of this complex, are accessory proteins that Cladosporium fulvum TTAGGG f
operate as a telomerase processing factor (Wang et al., 2007; Xin Glomus intraradices TTAGGG g
et al., 2007). In S. cerevisiae the evolutionarily conserved helicase Beauveria bassiana TTAGGG h
Pif1p regulates the amount of telomerase at chromosomal-ends Pestalotiopsis microspora TTAGGG i
Botrytis cinerea TTAGGG j
by displacing the enzyme from the ends while in Schizosaccharo-
Fusarium oxysporum TTAGGG k
myces pombe, Taz1 protein is required for DNA replication across Ustilago maydis TTAGGG l
the telomeres (Boule et al., 2005; Miller et al., 2006). Saccharomyces cerevisiae TG1–3 m
Adjacent to the telomeric repeats are the subtelomeric regions Schizosaccharomyces pombe TTAC(A)(C)G(1–8) n
that often consist of middle repeated sequences that may harbor Kluyveromyces lactis GGTGTACGGATTT GATTAGGTATGT o
Candida albicans GGTGTACGGATGT CTAACTTCTT p
genes. The chromatin structure of these regions has features of het-
Aspergillus oryzae TTAGGGTCAACA q
erochromatin and the potential to silence adjacent genes, a phe-
nomenon known as TPE (telomere position effect). Epigenetic (a) Bhattacharyya and Blackburn (1997), (b) Underwood et al. (1996), (c) Inglis et al.
(2005), (d) Farman and Leong (1995), (e) Schechtman (1990), (f) Coleman et al.
tags, such as histone hypoacetylation and DNA methylation of telo-
(1993), (g) Hijri et al. (2007), (h) Padmavathi et al. (2003), (i) Levis et al. (1997)and
meric and subtelomeric chromatin have also been observed; the Long et al. (1998), (j) Levis et al. (1997), (k) Powell and Kistler (1990), (l) Guzman
presence of such epigenetic tags correlates with telomere length and Sanchez (1994), (m) Shampay et al. (1984), (n) Matsumoto et al. (1987), (o)
regulation (Ottaviani et al., 2008). McEachern and Blackburn (1995) and McEachern and Hicks (1993), (p) McEachern
and Hicks (1993), (q) Kusumoto et al. (2003).
In contrast to the vast knowledge of telomere biology in model
organisms, our current information about the structure and func-
tion of telomeres in filamentous fungi is scarce. Ustilago maydis Adjacent to the telomere repeats are middle repetitive sequences
possess several advantages for genetic manipulation compared to that are usually found at several chromosomal ends and are highly
other fungal systems and has served as a model to study several polymorphic in length; these regions are commonly known as subt-
basic biological phenomena at the molecular level. The fact that elomeric regions or telomere-associated sequences (TAS). Saccharo-
it is able to grow as yeast-like cells and as hyphae and that fungal myces cerevisiae TAS harbors two main types of sequences known
mycelium can proliferate within tumors of maize tissue opens new as X and Y0 which may function as a buffer at chromosomal ends
opportunities to analyze telomere function during this peculiar (Zakian and Blanton, 1988). Saccharomyces cerevisiae telomerase
lifestyle. In this review we analyze structural features of the mutants undergo telomere attrition and senescence-related pheno-
telomeric region in U. maydis and other fungi and describe genes types. Survivor of telomerase mutants display rearrangements and
involved in well establish processes related to telomere function, amplifications within TAS, rendering chromosomes stable (Chen
correlating them with putative U. maydis homologs. This type of et al., 2001). Ustilago maydis TAS located adjacent to telomeric re-
data analysis through comparative genomics is useful to advance peats consist of two main basic classes of sequences, UTASa and
hypothesis aimed to find links between telomere function and UTASb. UTASa is highly conserved and is mostly located at chromo-
pathogenesis and/or development in fungi that can be later somal ends, whereas UTASb is less conserved and is found inter-
approached experimentally. spersed throughout the genome. Such sequences were identified
in five clones isolated from a chromosome end-enriched library of U.
2. Basic structural features of telomeric regions maydis DNA. UTASa encodes truncated versions of a helicase gene
(Sanchez-Alonso and Guzman, 1998).
The telomeric repeat in most eukaryotes consist of a short tan- In genome sequencing projects, telomere repeats and adjacent
demly repeated G-rich sequence. The G-rich strand protrudes as a TAS are rarely present or assembled within the chromosomal con-
single-strand tail whose length varies in a species-specific manner; tigs. Bioinformatic tools and direct cloning of chromosomal ends
in metazoans this single-stranded tail is usually longer and dis- are required to assemble the ends. TERMINUS (Telomeric End-Read
places the G-rich repeats at the double-stranded portion of the Mining IN Unassembled Sequences) is a tool that has been devel-
telomere. This arrangement is a half Holliday structure that con- oped to map telomere regions on complete genomes by assembling
tributes to telomere maintenance, and triggers the formation of a telomeric reads into chromosome end contigs (Li et al., 2005).
dynamically stable loop structure known as the T-loop (Griffith Using TERMINUS, 45 of the 46 U. maydis chromosome ends were
et al., 1999). In yeast, the T-loop is absent, nevertheless the telo- mapped; the assemblies of U. maydis telomere regions as well as
mere folds over subtelomeric regions, without invasion or base assemblies from other fungi are located at http://fungus.
pairing of DNA sequences; this folding is required for epigenetic kbrin.uky.edu/cgi-bin/gbrowse/U_maydis_broad_tel_100k.
control of regional silencing and telomere lengthening (de Bruin Magnaporthe oryzae is one of the few fungi to have all its chro-
et al., 2001). mosome ends sequenced from the telomeric repeats to the unique
In most filamentous fungi the basic telomere unit is 50 -TTAGG sequences within each chromosome (Rehmeyer et al., 2006). The
G-30 . This motif is conceivably the ancestral telomere repeat in fourteen chromosome ends were sequenced from fosmid clones
metazoans since lower metazoans such as corals, the group of containing telomeric DNA; three independent clones per end were
organisms that span the gap between the fungi and higher metazo- characterized. Eleven of the 14 ends contain highly conserved
ans, contain this repeat at their chromosomal ends (Sinclair et al., sequences encoding truncated versions of a helicase gene, one
2007; Traut et al., 2007). Indeed, this telomeric repeated hexamer chromosome end has rDNA connected to the telomere repeats,
is found in U. maydis and in filamentous fungi in general (see Table and in the two remaining chromosome ends, unique sequences
1). Telomere repeats show differences in some yeasts, the telomere are found adjacent to the telomere repeats. Transposon insertions
unit is longer in Candida sp. and Kluyveromyces lactis and shows an of various types are also common in M. oryzae chromosome ends
irregular repeat structure in S. cerevisiae and S. pombe (see Table 1). (Rehmeyer et al., 2006).
S56 P. Sánchez-Alonso, P. Guzman / Fungal Genetics and Biology 45 (2008) S54–S62
3. RecQ helicase genes linked to telomeres
A remarkable feature found at several fungal chromosomal ends
is the presence of highly conserved sequences that encode RecQ
helicases, located very close to the telomere repeats. Helicase
genes are generally found at S. cerevisiae chromosome ends. They
are encoded on the Y0 elements that become amplified in survivor
cells of telomerase negative mutants (Chen et al., 2001). In S. pom-
be sequences encoding RecQ helicases are located at the four chro-
mosome ends of two of its three chromosomes. Expression of such
helicases increases in survivor populations of telomerase mutants.
Likewise, overexpression of a helicase domain in S. pombe telome-
rase negative mutants allows a quicker recovery after crisis, sug-
gesting a role in telomere maintenance under this particular
condition (Mandell et al., 2005).
RecQ helicase genes associated with chromosome ends were
first described in U. maydis (Sanchez-Alonso and Guzman, 1998).
They were then found in a similar location in M. grisea (Gao
et al., 2002), in M. oryzae (Rehmeyer et al., 2006), Metarhizium ani-
sopliae (Inglis et al., 2005) and S. pombe (Mandell et al., 2005).
Apart from the work of telomere-linked RecQ helicases in S. pombe
and the distant helicase encoded in the Y0 element of S. cerevisiae,
little is known about the role of such sequences. The location of
RecQ helicase genes close to the chromosome ends may corre-
spond to a primitive mechanism for chromosomal stabilization in
the absence of telomerase. A meaningful correlation is the fact that
WRN, a protein responsible for the Werner Syndrome in humans
and Sgs1p in S. cerevisiae encode RecQ helicases. Although, WRN
and Sgs1p are not telomere-linked, they are required for telomere
metabolism. WRN interacts with TRF1, TRF2 and POT1 that are
components of the telosome/shelterin complex and Ku70/80 a het-
erodimer which has a role in telomere maintenance (Multani and
Chang, 2007).
4. Gene families encoded at chromosome ends
In several fungi and protists genes encoding proteins involved
in interactions with the environment often increase in number
near telomeres. These genes, termed ‘contingency genes’, often dis-
play allelic diversity and in most cases are dispensable for survival,
nevertheless they grant possibilities to confront new environ-
ments. In the human parasites Plasmodium falciparum and Trypan-
osoma brucei, families of antigenic surface proteins that induce the
immune system response are encoded in telomeric regions. This is
proposed to be part of a mechanism developed to amplify and
diversify surface antigen genes which may hinder host recognition
(Barry et al., 2003).
Saccharomyces cerevisiae, depending on its niche, encodes genes
near telomeres that display variation in gene amplification and/or
expression. Some of these are the PAU genes that represent the
largest multigene family in S. cerevisiae with 23 members, most
of them located near the telomeres. PAU genes are regulated by
anaerobiosis, and expression analysis showed that some are in-
duced during alcoholic fermentation and are negatively regulated
by oxygen (Rachidi et al., 2000). Others are the MAL and MEL genes
that are required for maltose and alpha-galactosidase fermenta-
tion, respectively, and are advantageous in strains used in the bak-
ing and brewing industries (Gibson et al., 1997; Teunissen and
Steensma, 1995). Likewise, the FLO genes that encode cell-wall gly-
coproteins participate in the regulation of cellular adhesion (Halme
et al., 2004).
The gene content at chromosome ends has also been examined
in other fungi. The TLO family of C. albicans consists of 15 members,
14 of which are located at chromosome ends. This family is unique
in C. albicans and members are present on every chromosome.
The function of these genes is unknown, although a member of this
family was identified as a putative transcription factor in a yeast
one-hybrid screen (van het Hoog et al., 2007). In K. lactis 30% of
genes located at chromosome ends encode proteins predicted to
be extracellular or plasma membrane proteins. Other genes encode
FLO-like proteins, which also show a telomere-associated location
in S. cerevisiae (Fairhead and Dujon, 2006). Pathogenic fungi may
encode families of well characterized or putative virulence factors
at chromosome ends. Almost two dozen genes encoding adhesins
are located at telomeric regions in Candida glabrata (De Las Peñas
et al., 2003). Likewise, in Pneumocystis carinii clusters of major sur-
face antigen genes are predicted to occur at every chromosome end
(Keely et al., 2005).
Little is known about the presence of contingency genes or
avirulence genes at chromosome ends of phytopathogenic fungi,
nevertheless genes with avirulence function in some pathogenic
isolates, have been found near telomere ends (Farman, 2007).
Although this observation hints at the possibility that phytopatho-
genic fungi can use a strategy to amplify genes participating in host
evasion at chromosome termini, the number of avirulence genes in
this location is not significantly represented (Farman, 2007). In U.
maydis two gene families associated with chromosome ends has
been predicted (Kamper et al., 2006). Most members of this family
are located within 14 kb at the ends of the chromosomal contigs
(see Fig. 1). The function of such families is unknown, although
they are known to be Ustilago-specific genes, mostly pseudogenes,
displaying a high degree of conservation between them (Kamper
et al., 2006).
5. Telomerase components in Ustilago maydis
5.1. Reverse transcriptase catalytic subunit
The central components of the telomerase enzyme are the re-
verse transcriptase catalytic subunit (TERT) and the RNA template
that is associated non-covalently to the catalytic subunit (Greider
and Blackburn, 1989). Additional protein components include
EST1a and EST1b and the dyskerin (dyskeratosis congenital) in
mammals, and Est1, Est3 and SM7 proteins in S. cerevisiae (Smog-
orzewska and de Lange, 2004). The TERT subunit contains at least
four domains. The first is RT, comprising seven conserved motifs (1,
2, A, B0 , C, D, E), that correspond to the reverse transcriptase
domain which catalyzes the synthesis of the G-rich strand from
the RNA template moiety; RT is present in all characterized TERTs.
Next, is the RNA-binding domain that spans three conserved
motifs, CP, QFP, and T, and is required for specific binding of TERT
to the telomerase RNA subunit. The third domain, the N-terminal
domain, may have a conserved region known as GQ which is only
needed for in vivo activity. Finally, the C terminal domain which
appears to be involved in processivity and recruitment of the telo-
merase to the telomere (Autexier and Lue, 2006). Gene UM00761.1
is likely to encode the catalytic core of the U. maydis telomerase
since it contains these four conserved domains including all the
motifs present in known TERTs (Sanchez-Alonso et al., unpub-
lished). Fig. 2A shows a phylogeny comparing known TERTs and
possible homologs obtained from fungal genomes following BLAST
analysis at available databases. Ustilago maydis TERT is found in the
same clade as predicted TERTs from other filamentous fungi, and
more specifically shares a branch with Coprinopsis cinerea, another
basidiomycete.
5.2. Dyskerin
Dyskerin is basically, a small-nucleolar ribonucleoprotein
(snoRNP) with pseudouridine synthase activity that associates
 P. Sánchez-Alonso, P. Guzman / Fungal Genetics and Biology 45 (2008) S54–S62 S57

Fig. 1. Location of two telomere-associated protein families in U. maydis. Boxes represent left and right ends from the assembled chromosomes where members of Families
#1 and #2 are encoded. ORFs were taken from previously inferred coordinates (see Supplementary Table S2 in Kamper et al. (2006). They are displayed as black boxes for
Family #1 and gray boxes for Family #2. Beginning at the chromosome ends, a 1kb scale is shown at the bottom of the Figure.

Fig. 2. Phylogenetic trees of (A) TERT and (B) Dyskerin proteins. Each tree includes representative proteins from fungi, plants, animals and protists. (A) The phylogenetic tree
is based on the alignment for T, 1, 2, A, B0 , C, D, and E motifs from the RNA-binding and the RT domains of the TERT catalytic core. Accession numbers are as follows:
Saccharomyces cerevisiae (NP_013422.1), Candida albicans (XP_717626.1), Schizosaccharomyces pombe (AAC49802), Ustilago maydis (XP_756908.1), Coprinopsis cinerea
(EAU85360.1), Cryptococcus neoformans (XP_775503.1), Botryotinia fuckeliana (XP_001561236.1), Magnaporthe grisea (XP_363691.1), Gibberella zeae (XP_381218.1), Aspergillus
fumigatus (XP_749051), Aspergillus clavatus (XP_001273297.1), Zea mays (AAX21528.1), Oryza sativa (AAK35007.1), Arabidopsis thaliana (NP_197187.1), Vitis vinifera
(CAO40853.1), Populus trichocarpa (gw1.III.1219.1), Xenopus laevis (AF212299_1), Mus musculus (NP_033380.1), Canis familiaris (NP_001026800.1), Homo sapiens
(NP_937983.2), Tetrahymena termophila (O77448), Euplotes aediculatus (O00939), Oxytricha trifallax (O76332). (B) Phylogenetic tree is based on TrueB_N/PUA pseudouridine
synthases similar to DKC or Cbf5. Sequences were delimited to the three conserved overlapping-regions of dyskerin: the DKCLD N-terminal domain, tRNA pseudouridine
synthases B region, and human dyskerin-like pseudouridine synthase region. Accession numbers are as follows: Ustilago maydis (XP_756832.1), Coprinopsis cinerea
(EAU92842.1), Criptococcus neoformans (XP_570199), Emericella nidulans (O43100.1), Gibberella zeae (XP_387382), Magnaporthe grisea (XP_367607.2), Saccharomyces cerev-
isiae (P33322), Candida albicans (O43101), Schizosaccharomyces pombe (O14007), Arabidopsis thaliana (Q9LD90), Vitis vinifera (CAO15636), Hordeum vulgare (CAB85492), Oryza
sativa (AAG46137), Chlamydomonas reinhardtii (EDP05982), Homo sapiens (NP_001354), Canis familiaris (XP_549382), Mus musculus (NP_001025478), Gallus gallus
(NP_001026286), Xenopus laevis (AAI30043), Drosophila melanogaster (AAD19897), Caenorhabdithis elegans (O17919), Tetrahymena termophila (XP_001012103). Trees were
created using the neighbor-joining method with a bootstrap value of 1000 replicates and plotted using NJplot.
S58 P. Sánchez-Alonso, P. Guzman / Fungal Genetics and Biology 45 (2008) S54–S62

early with the telomerase catalytic core during the assembly of
TERT with the RNA template. Dyskerin, comprises several proteins
that along with the template RNA form a complex (dyskerin
complex) involved in telomerase performance and stability
(Smogorzewska and de Lange, 2004). Mass spectrometric sequenc-
ing-analysis of purified human telomerase indicates that the telo-
merase consists of telomerase reverse transcriptase, telomerase
RNA, and dyskerin (Cohen et al., 2007). In mammals, the Dyskerin
complex includes four proteins, NAP57/Dyskerin, NOP10, NHP2
and GAR1. These are encoded by highly conserved genes in eukary-
otes. In S. cerevisiae, the four possible components are Gar1p, Cbf5p
(the ortholog of dyskerin), Nhp2p, and Nop10p (Henras et al.,
2004). Fig. 2B shows a phylogeny comparing known dyskerins
and possible homologs obtained from several genomes including
fungi after BLAST searches. The probable dyskerin gene in U. may-
dis is UM00685.1, and it is found in the same clade as other
predicted fungal dyskerins, particularly with dyskerins from the
basidiomycetes C. cinerea and Cryptococcus (Sanchez-Alonso
et al., unpublished).
5.3. Est1 protein
A third component of the telomerase is the Est1 protein. Est1 is
a non-catalytic protein required for proper telomerase function
(Lendvay et al., 1996). In S. cerevisiae, Est1 assists in the recruit-
ment of the telomerase to short chromosome termini which are
preferred substrates for this enzyme (Bianchi and Shore, 2007).
Est1 is also required for telomerase assembly and activation in
Candida albicans (Hsu et al., 2007). In contrast to the other telome-
rase components, sequence conservation of Est1 is limited among
eukaryotes. BLAST sequence searches in databases identified the
locus UM04177.1 as the putative U. maydis Est1 gene (Sanchez-
Alonso et al., unpublished).
6. Predicted U. maydis telomere-associated proteins
Telomere-associated proteins define another class of compo-
nents of telomeres. These proteins are essential for the organiza-
tion and homeostasis of the ribonucleoprotein structure of the
telomere. Telomere-associated proteins have important functions
in processes such as the formation of the telomere cap, replication
control by the telomerase pathway, alternative lengthening based
on homologous recombination and in nuclear membrane binding,
among others. Several of the telomere-associated proteins are con-
served among eukaryotes (Smogorzewska and de Lange, 2004). We
searched the U. maydis predicted proteome for possible homologs
of conserved telomere-associated proteins. Table 2. lists the pro-
teins that were found along with the predicted U. maydis genes.
6.1. Telosome/shelterin complex components
Telomere maintenance relies on a multi-subunit complex
known as telosome/shelterin. This complex is formed by proteins
with related functions in metazoan and unicellular eukaryotes.
The function and location of the telosome/shelterin complex is re-
stricted to the chromosome termini (Smogorzewska and de Lange,

Table 2
Predicted loci for telomere-associated proteins in Ustilago maydis

Ustilago maydis locus Telomere-associated Best alignment hit to Score and E-value Effect on telomeres Refs.
protein currently characterized
homologs (accession)
UM05117.1 POT1 Gallus gallus POT1 homolog S = 48.5 E = 5.6  106 Components of the telosome/shelterin a, b
(NP_996875) complex. Protection of telomere, telomere
UM02326.1 TRF1 Muntiacus reevesi TRF1 S = 55.8 E = 2  105 length regulation
(AAU43270.1)
UM05756.1 Ku80 Aspergillus sojae Ku80 S = 207 E = 2  1051 Components of the Ku DNA-damage response c
(BAE78504) heterodimer. Protection at telomere,
UM05148.1 Ku70 Aspergillus oryzae Ku70 S = 225 E = 1  1056 recognition and binding of DNA DBS. Involved
(BAE78501) in NHEJ recombination
UM01085.1 Rad50 Mus musculus Rad50 S = 501 E = 1  10139 Components of the MRN/MRX complex. d, e
(NP_033038) Generation of G-overhangs. Prevention of re-
UM04704.1 Mre11 Coprinopsis cinerea Mre11 S = 575 E=4  10162 replication. Required for ALT mechanism
homolog (Q9UVN9)
UM01003.1 Nbs1 Mus musculus Nbs1 S = 52.8 E = 2  104
(NP_038780)
UM02874.1 RecQ-like helicases Schizosaccharomyces pombe S = 567 E = 2  10159 Maintaining genomic stability. Resolution of f, g, h
Rqh1 (NP_593092.1) telomere recombination intermediates where
replication stall, and at an interface between
DNA replication and DNA repair in
dysfunctional telomeres
UM01110.1 ATR, ATM Homo sapiens ATR S = 806 E = 0.0 Signal transducer, and component of telomere i, j, k
(CAA70298) capping functions Controls cellular response to
DSB
UM00963.1 SIR2family of proteins Mus musculus Sirt1 S = 280 E = 2  1073 Histone deacetylase. Spreading of silencing l
(NP_062786.1)
79
UM058920.1 Gallus gallus Sirt1 S = 297 E = 9  10
(NP_001017414)
UM06360.1 Nph2 Homo sapiens Nph2 S = 183 E = 3  1045 Dyskerin complex components along with m, n
(NP_004999) DKC1. Involved in telomerase catalytic core
UM04573.1 Gar1 Homo sapiens Gar1 S = 101 E = 3  1020 and RNP biogenesis
(NP_061856)
UM03354.1 Nop1 Saccharomyces cerevisiae S = 901 E = 4  1017
Nop1 (2AQA_A)

Protein BLASTs were performed by using BLOSUM62 matrix and default gap costs. (a) Baumann and Cech (2001); (b) de Lange (2005); (c) Ribes-Zamora et al. (2007); (d) Chai
et al. (2006); (e) Larrivee et al. (2004); (f) Opresko et al. (2004), (g) Li et al. (2004); (h) Eller et al. (2006); (i) Lavin and Kozlov (2007), (j) Sabourin et al. (2007); (k) Hector et al.
(2007); (l) Kaeberlein (2007); (m) Cohen et al. (2007), (n) Henras et al. (2004).
 P. Sánchez-Alonso, P. Guzman / Fungal Genetics and Biology 45 (2008) S54–S62
2004). These proteins have important roles, such as determining
the structure and stability of T-loops, regulating the access of telo-
merase to telomeric ssDNA and safeguarding chromosome ends to
prevent recognition as double-strand breaks (Griffith et al., 1999).
The yeast telosome complex includes the Rap1 and Cdc13 proteins,
which interact directly with double-stranded and single-stranded
telomeric DNA, respectively, and also factors recruited by Rap1.
Those factors are the silent information regulators Sir2, Sir3 and
Sir4, and the RAP1 interacting proteins Rif1 and Rif2. In humans
the shelterin complex consists of six proteins: POT1, TRF1, TRF2,
TIN1, Rap1 and TPP1 (Blasco, 2007; de Lange, 2005). Information
on the functions of these components and their presence in U. may-
dis is presented below.
6.1.1. Protection of the chromosome termini
A key component required for the protection of chromosome
ends and the regulation of telomere length in S. pombe and mam-
mals is POT1 (Protection Of Telomeres) (Baumann and Cech, 2001).
POT1 is a single-stranded telomere DNA binding protein ortholo-
gous to yeast Cdc13. Cdc13 is enriched at the single-stranded do-
main of telomeres at late S-phase together with the essential
endonuclease Dna2, which is thought to generate the long TG-rich
single-strand overhangs of telomeric DNA that are also present at
late S-phase (Tomita et al., 2004). Cdc13 recruits telomerase possi-
bly through a direct interaction with Est1 (Bianchi et al., 2004). In
humans, Pot1 interacts with TIN2, and with TPP1, the homolog of
ciliate TEBP beta (Xin et al., 2007). TPP1 regulates the levels of
POT1 and TIN2 both within the nucleus and in the cytoplasm, since
it contains a functional nuclear export signal, which aids nuclear
traffic of these shelterin complex proteins (Chen et al., 2007).
TIN2 protects TRF1 from ADP-ribosylation by tankyrase (a poly-
adenosine diphosphate–ribose polymerase). Poly-ADP ribosylation
of TRF1 leads to its dissociation of double-strand telomeric DNA
and destruction by ubiquitination mediated proteolysis (Ye and
de Lange, 2004). POT1 is highly conserved throughout evolution
and candidate genes can be predicted in several organisms from
mammals and plants to unicellular eukaryotes, whereas TPP1
and TIN2 are much less well conserved and cannot be readily iden-
tified in available databases (Baumann and Cech, 2001).
Of the six proteins of the shelterin complex, TRF1 and TRF2 rec-
ognize and bind to double-stranded telomeric repeats. Similar to
Rap1 from S. cerevisiae, and TAZ1 from S. pombe, TRF1 and TRF2
have a SANT/myb-like domain that recognizes double-stranded
telomere sequences. This binding is essential for telomere length
control. In mammalian cells TRF2 interacts with POT1 and with
hRAP1 (Hug and Lingner, 2006). On the other hand, TRF1, is a neg-
ative telomere-length regulator, and its binding to the double-
strand telomere repeat is controlled by tankyrases 1 and 2 and
by TIN2. Access of telomerase could be possible through the disso-
ciation and ubiquitin-mediated proteolysis of TRF1 (Chang et al.,
2003). Mutations in either Rap1, TAZ1, TRF1 or TRF2 lead to a telo-
mere elongation phenotype (Hug and Lingner, 2006). A possible
homolog of TRF is also predicted in U. maydis (see Table 2).
6.1.2. Epigenetic silencing at chromosome termini
A conserved feature of telomeres is the heterochromatization of
telomere-associated regions that results in a silencing effect
known as telomere position effect or TPE. Silencing is a reversible
state of transcriptional repression that initiates with heterochro-
matization at specific sequences and then expands to adjacent
regions. This function of telomeres is essential for chromosome
maintenance. The telosome/shelterin complex and the siRNA
machinery mediate telomeric silencing (Blasco, 2007; Smog-
orzewska and de Lange, 2004). In S. cerevisiae, Sir2/Sir3 complex
is recruited by Rap1 or Ku to the telomere. Sir2, a histone deacetyl-
ase type III, removes acetyl residues from histones to spread the
S59
silenced chromatin (Kaeberlein, 2007). Sir2-related proteins are
broadly named sirtuins (North and Verdin, 2004). A potential
homolog of Sir2 is predicted in U. maydis (see Table 2).
Telomere length has a major effect on the epigenetic status of
chromosome ends. Acetylation and methylation of histones are
part of the mechanism to regulate the length of telomere repeats
which is important for maintenance of telomere architecture. Loss
of telomeric repeats lead to a loss of heterochromatic architecture
at chromosome termini. Thus, histone modifications are required
to maintain a stable chromatin structure and for the counting of
telomere repeats (Blasco, 2007).
6.2. DNA-damage response
DNA double-strand breaks (DSBs) are recognized by DNA-dam-
age response (DDR) factors. A basic role of telomeres is to prevent
chromosome ends from being recognized as DSB. Mre11/Rad50/
Nbs1 (MRN) in mammals and Mre11/Rad50/Xrs2 (MRX) in S. cere-
visiae are complexes involved early in DSB repair. They are widely
conserved in evolution, and their components have been reported
in many metazoans and unicellular eukaryotes (Chai et al., 2006;
Larrivee et al., 2004). MRN/MRX also function in S-phase check-
point activation, in prevention of DNA re-replication and in the
maintenance of telomere integrity (Lee et al., 2007; Olson et al.,
2007). Indeed several functions for MRN/MRX at telomeres have
been described. In mammalian cells, after conventional DNA syn-
thesis, telomeric DNA resembles DSB and MRN/MRX complexes re-
cruited by TRF2 participate in the generation of 30 single-strand
overhangs by C-strand resection at the S-phase of the cell cycle
(Zhu et al., 2000). In yeast, knock-out of MRX components resulted
in a shortening of G-overhangs (Larrivee et al., 2004; Zhong et al.,
2007) and depletion of the human MRN complex, affects the route
of alternate lengthening (Larrivee et al., 2004; Zhong et al., 2007).
The MRE/MRX complex is likely to be present in U. maydis since
homologs of Mre11, Rad50 and Nbs1 are predicted to be encoded
in the genome (see Table 2).
Members of the RecQ family of DNA helicases participate in
telomere metabolism by interacting with proteins involved in the
maintenance of the chromosome termini as well as by setting up
of the DNA-damage response at the telomere. The RecQ family of
DNA helicases comprises a highly conserved group of enzymes that
exhibits homology with, and receives its name from, the RecQ heli-
case of Escherichia coli (Irino et al., 1986). Mutations in RecQ heli-
case genes cause genome instability, hyper-recombination, and
hypersensitivity to DNA-damaging agents. In S. cerevisiae the
recombination based ALT pathway require the RecQ homolog
Sgs1p to allow Type II survivors to rise in telomerase-negative mu-
tants (Huang et al., 2001; Johnson et al., 2001). In humans there are
five helicase RecQ homologs, mutations in three of them trigger
syndromes associated with genome instability and cancer (Opresko
et al., 2004). Such is the case of Werner (WRN) that initiates the
DNA-damage response pathway when telomeres are disrupted
(Eller et al., 2006). In addition WRN interacts with TRF1, TRF2
and POT1, components of the shelterin complex, and with the Ku
complex (Li et al., 2004; Opresko et al., 2004). The Ku complex is
a DNA-damage response heterodimer that has a dual function of
recognizing and protecting the chromosome termini and recogniz-
ing non-homologous-end-joining (NHEJ) when DNA double-strand
breaks occurred. The dual activities of the Ku heterodimer, which
consists of Ku70 and Ku80, relies in the different affinities defined
by alpha helices located on each surface of the two sides of the
complex. Ku70 binds to the DNA end, which is required for NHEJ,
and Ku80 binds to the telomere and is found in the telomere het-
erochromatin (Ribes-Zamora et al., 2007). Homologs of a WRN
RecQ helicase and the Ku complex (Ku70 and Ku80) may be pres-
ent in the U. maydis genome (see Table 2).
S60 P. Sánchez-Alonso, P. Guzman / Fungal Genetics and Biology 45 (2008) S54–S62

The ATM protein kinases family also has an effect on telo-
mere homeostasis. ATM and ATR kinases are members of the
phosphatidyl inositol 3-kinase-like family of signal transducers
that are conserved among living organisms. The human ATM
(ataxia telangiectasia mutated) and ATR (ataxia telangiectasia re-
lated) kinases and their yeast homologs Tel1p and Mec1p ki-
nases respectively, function upstream in the checkpoint
pathway for DNA damage repair response (Lavin and Kozlov,
2007). They are positive telomere length regulators that act in
response to telomere shortening. Tel1p preferentially associates
to short telomeres and this association is stimulated by Mre11
(Hector et al., 2007; Sabourin et al., 2007). A substrate of Tel1p
and Mec1p is the single-strand binding protein Cdc13. Thus,
phosphorylation of Cdc13p together with the MRX complex are
important for the maintenance of chromosome termini regulat-
ing telomerase recruitment in the telomere (Tseng et al., 2006).
A locus with strong similarity to ATM kinases is present in the
U. maydis genome (see Table 2).
7. Closing remarks
Telomeres and telomere-associated sequences are active
regions required not only for maintenance of chromosomal integ-
rity but often also as a reservoir of genetic information valuable
for environmental adaptation. In this review, we have summarized
general features of telomere biology and suggested candidate U.
maydis genes that are potentially involved in telomere metabolism.
Although, genome sequencing projects are invaluable resources
that reveal the DNA sequence at the chromosome ends, sequences
at the tip of the chromosome are often not present within the chro-
mosomal contigs. Furthermore, telomeric regions are enriched for
DNA repetitive elements that make the assembly of the sequences

Fig. 3. A model for telomere organization and function in U. maydis. The structure and associated components of the chromosome ends from (A) S. cerevisiae, (B) S. pombe and
(C) U. maydis are shown. (A) and (B) are based on published findings (Blasco, 2007; Eller et al., 2006; Hug and Lingner, 2006; Lavin and Kozlov, 2007; Lee et al., 2007); in (C)
the model was deduced based on the predicted proteins shown in Table 2.
 P. Sánchez-Alonso, P. Guzman / Fungal Genetics and Biology 45 (2008) S54–S62
a difficult task. Thus, to have an accurate vision of the chromosome
ends it is often necessary to make use of strategies to directly clone
the tip of the chromosome up to the single copy sequences. M. ory-
zae is the only filamentous fungus to have all its chromosome ends
revealed. A common feature found adjacent to the telomere re-
peats is the occurrence of sequences encoding RecQ helicases.
Complete and truncated versions of RecQ helicases are often pres-
ent at several chromosome ends. This feature may be unique to
fungal genomes and has been detected in 10 additional fungal gen-
omes. Species-specific genes are also common at chromosome
ends. Indeed, U. maydis encodes two such gene families (see
Fig. 1). The identity and function of such genes has not been estab-
lished and a comparative analysis in various independent U. maydis
isolates may be useful to understand the nature of these gene
families.
Bioinformatic analysis revealed potential U. maydis homologs
involved in telomere metabolism. Two genes of the telomerase
complex were readily identified, genes encoding the catalytic sub-
unit and dyskerin, suggesting that the mechanism previously de-
scribed in model systems for telomere synthesis is conserved in
U. maydis. Likewise, genes encoding the telomere-associated pro-
teins POT1 and TRF1 or the MRN/MRX and Ku complexes are likely
to be conserved in U. maydis. These findings indicate that the
essential functions of the telomere, as well as the recognition of
DNA double-strand breaks by the DNA damage repair complex,
are also conserved in U. maydis. A model for telomere organization
and function in this fungus is shown in Fig. 3.
In summary, little information has been generated on the struc-
ture and function of telomeres in filamentous fungi. Complete
analysis of full chromosome termini lies ahead for fungal genomes.
The assumption that the telomere-associated region is an impor-
tant reservoir of gene families encoding contingency-like genes
must be confirmed. Likewise, the role of the telomerase complex
during the cell cycle and the life cycle of fungi and particularly in
dimorphic fungi will have to be assessed.
Acknowledgments
We thank June K. Simpson and Gabriela Olmedo for reviews and
comments on the manuscript.
This work is supported by grants from CONACyT, Mexico.
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