At the Beginning of the End and in the Middle of the Beginning: Structure and Maintenance of Telomeric DNA Repeats and Interstitial Telomeric Sequences

Abstract

Tandem DNA repeats derived from the ancestral (TTAGGG)n run were first detected at chromosome ends of the majority of living organisms, hence the name telomeric DNA repeats. Subsequently, it has become clear that telomeric motifs are also present within chromosomes, and they were suitably called interstitial telomeric sequences (ITSs). It is well known that telomeric DNA repeats play a key role in chromosome stability, preventing end-to-end fusions and precluding the recurrent DNA loss during replication. Recent data suggest that ITSs are also important genomic elements as they confer its karyotype plasticity. In fact, ITSs appeared to be among the most unstable microsatellite sequences as they are highly length polymorphic and can trigger chromosomal fragility and gross chromosomal rearrangements. Importantly, mechanisms responsible for their instability appear to be similar to the mechanisms that maintain the length of genuine telomeres. This review compares the mechanisms of maintenance and dynamic properties of telomeric repeats and ITSs and discusses the implications of these dynamics on genome stability.

Keywords: alternative lengthening of telomeres; genome stability; microsatellites; repeat expansion; telomeres; telomeric repeats.

Figure 1

Abridged scheme of telomere organization in vertebrates and in budding yeast showing major telomere-specific protein. (A) Schematic representation of the shelterin complex and CST-complex bound to a vertebrate telomere. TRF1 and TRF2 proteins bind to the double-stranded telomeric DNA, POT1/TPP1 and CST-complex bind to the G-rich single-stranded telomeric overhang. Invasion of the G-rich overhang into the double-stranded telomeric repeat transforms this protein-DNA complex into a lariat-like structure called a T-loop. Nucleosomes and secondary protein factors implicated in telomere maintenance are not shown. (B) Proteins specific to yeast telomeric repeats include Rap1 which binds to the double-stranded telomeric repeat and recruits Rif1 and Rif2 or Sir-proteins, and CST-complex bound to the G-rich single-stranded overhang. The Tbf1 protein binds to human telomeric repeats, called STAR-repeats, located at subtelomeric DNA regions. Yeast telomeres can also form a folded-back structure, called telosome, which is stabilized by protein-protein interactions. Sirtuins and Yku70/Yku80 heterodimer participate in telosome formation and telomere maintenance in yeast.

Figure 2

Telomere length maintenance mechanisms. (A) A simplified view of telomere length control by telomerase and the CST-complex. Telomerase adds telomeric repeats to the 3’-end of the G-rich overhang. Its activity is controlled by the CST-complex, which displaces telomerase, removes secondary structures and recruits the DNA polymerase α/primase complex to synthesize the C-rich strand. Note that regulation of telomerase by the CST-complex is more complex in yeast Saccharomyces cerevisiae (see text for details). (B) A general representation of the Alternative Lengthening of Telomeres (ALT) mechanism. ALT is currently viewed as the BIR-dependent elongation of telomeres after the replication fork stalling, ultimately leading to the formation of a one-ended double-strand break. This fork stalling can be due to the formation of unusual secondary structures in telomeric DNA (such as G-quadruplexes), the presence of stable DNA-RNA structures (R-loops generated during transcription of telomere-specific RNA called TERRA) or potent protein barriers (a circle with question mark). The replicative helicase is pictured as a red ring, RNA polymerase is a yellow oval and DNA polymerases are green and blue torpedoes with rings on their rear ends representing PCNA. One-ended DSBs are then channeled into the RAD52-dependent DNA damage repair pathway. This pathway is either RAD51-dependent, which involves invasion into a homologous or homeologous duplex, or RAD51-independent, which involves single-strand annealing. (These two pathways lead to the formation of Type I and Type II survivors in telomerase-deficient S. cerevisiae, respectively.) Upon template switching, the replisome is assembled and BIR-dependent synthesis of the telomere proceeds. BIR at telomeres requires DNA polymerase δ, its accessory subunit POLD3 (Pol32 in S. cerevisiae) as well as Replication Factor C (RFC) and PCNA (see text for details). The source of templates for RAD51-independent annealing is not limited to single-stranded regions released after G-quadruplex formation in the complimentary strand. For a comprehensive review of all eligible substrates see [50]. Importantly, such templates may be represented by extrachromosomal telomeric circular DNAs believed to be generated from the resolution of recombination intermediates at telomeres. Other sources for template DNA during BIR-dependent telomere-lengthening could be T-loops or interstitial telomeric sequences (ITSs) (for further discussion of these issues see Section 2.4).

Figure 3

Mechanisms of Interstitial Telomeric Sequences (ITSs) instability (based on yeast model). (A) ITSs represent polar blocks for replication machinery [123,299,300]. Factors binding specifically to ITSs sequences (such as Rap1 in yeast, yellow stones on the figure, “protein roadblock” sign) influence the replication passage through this sequence depending on the orientation of the ITS tract. Additional factors interacting with Rap1, such as Sir-complex or Rif1 and Rif2 proteins (brown and gray stones), may enhance the stalling of the replication fork and regulate the replication, transcription and repair processes at ITS. Replication stalling is facilitated by the replication fork pausing complex component (Tof1-Csm3 in yeast, “stop,” “end freeway” signs) and can lead to single-stranded gap (ss-gap) formation. The stalled replication forks and ss-gaps can be repaired via HR (“recombination exit” sign) or postreplication DNA repair (“repair exit” sign). Template Switching (TS) (“detour” sign) is essential for both pathways. Replication from the other direction can result in replication fork collapse and DSB formation (“end of road” sign). In this case the gross chromosomal rearrangements can be formed. (B) The yeast model system we used to study ITS. The URA3 reporter gene, which is commonly used for both direct and counter-selection, is split with an artificial intron carrying the insertion of the telomeric tract. The UR-Int(YTEL)-A3 reporter cassette is placed in the chromosome III near ARS306. Placing telomeric tracts of varying length in different orientations and selection for the reporter gene inactivation events (on 5-FOA media) or reporter gene activation events (Ura- media) allows us to select for different events induced by the ITS tract as well as the length of the ITS tract. (C) The overview of the events induced by the ITS tract depending on its orientation. G- and C-rich strand denote the lagging strand template for replication and sense strand for transcription.

Figure 4

Possible roles of ITSs in the genome. (A) ITSs can participate in the formation of the Interstitial t-loop (ITL). This ITL can play an essential role in telomere maintenance and regulation of gene expression. Mediated by Lamin A/C and TRF2, the interaction of the ITS with the telomere can regulate the chromosome structure and position into the nucleus. Juxtaposion of the telomere and ITS may influence expression of genes near the ITS [267,297,326,329,330]. (B) ITSs can serve as boundary elements in the genome. Through recruitment of proteins that specifically bind to telomeric repeats and possess general regulatory activity, such as Rap1 in yeast [109] (yellow oval), ITSs can regulate the structure of adjacent chromatin and divide the chromosome into subdomains (orange and green rectangles). A possible interaction of ITSs with the nuclear lamina may regulate the 3D genome structure. (C) ITSs are potent elements conferring karyotype plasticity. ITSs can serve as spare sites for telomere formation and can impact the outcome of DSB repair [335] and can seed a new telomere. (D) Recombination between the ITS tract and the telomere can result in the inversion of the chromosome arm with a consequent effect on gene expression [299,336].

Figure 5

Alternative lengthening of telomeres and ITSs. (A) ALT is a process that relies on Template Switching (TS) and is thought to be initiated by replication blockage or a break in the DNA. The strand exchange reaction performed by specialized enzymes (e.g., TS helicases) or HR-proteins is followed by the DNA synthesis performed in BIR-like manner. The source for the template could be either the sister telomere or a telomere on another chromosome. (B) Extension mechanism via intratelomere invasion is also discussed [50,228]. (C) Another striking possibility is the usage of ITSs for telomere extension. In this case, an Interstitial t-loop (ITL) should be formed. The extension might occur via multiple cycles of invasion or DNA slippage. (D) Key components participating in ITS expansion are relevant for the ALT process [300], hence these two processes share certain similarities. An exciting prediction from this model is that ITS’s length destabilization can be an indicator of activated ALT.