Understanding how single-stranded DNA binding proteins acting at telomeres deal with the DNA secondary structures that can form at telomeres

In eukaryotes where telomeres are elongated by telomerase, the telomeric DNA strand running toward the 3’ end (“G-strand”) is generally composed of repeats of a short motif of 5–8 nucleotides (“telomeric motif”) carrying 2, 3 or 4 consecutive guanines and, in a variety of eukaryotes, it ends with a 3’ single-stranded overhang (“G-overhang”). The human telomeric motif is the hexamer GGGTTA. The GGGTTA motif has first been identified in human telomeres; it is conserved in vertebrates and is found in many other eukaryotes. The presence of consecutive guanines makes the telomeric G-strand prone to fold into G-quadruplexes (G4) (doi: 10.1093/nar/gkq1292).

The projects developed in our team in the past years were aimed at unravelling the structures formed by long telomeric sequences and understanding how single-stranded DNA binding proteins acting at telomeres interact with these DNA structures. Here below, we present our main achievements in this field of investigation.

Structure and stability of long GGGTTA repeats. The telomeric G-strand can form contiguous G4, like beads-on-a-string, that we characterised in two studies we published in 2015 and 2016. We showed that a sequence composed of a number of GGGTTA multiple of 4 forms in potassium contiguous G4 units that do not interact with each other and have similar stabilities, independently of the number of G4 units (doi: 10.1016/j.biochi.2015.04.003).

Contiguous G4 units

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©P. Alberti

Overall our study show that contiguous G4s formed by GGGTTA repeats exhibit at least two characteristics that make them easily manageable structures compared to other G4s: (i) they are stable, but not too stable; (ii) they are independent (not interacting with each other) and identical (similar stability) structural units. As we will see below, these features make telomeric contiguous G4 easily manageable structures for proteins that have to unfold them and affect the way these proteins deal with them. 

The folding into independent and identical G4 units is characteristic of telomeric repeats in potassium. Nevertheless, we highlighted an exception that confirm this rule: an original behaviour where two contiguous telomeric G4 (and only two) in sodium (and only in sodium) interact with each other forming a very stable higher-order structure  (doi: 10.1093/nar/gkw003).

How do single-stranded DNA binding proteins deal with telomeric G4? At telomeres, contiguous G4 can form at 3’ single-stranded overhang (G-overhang) as well as in the double-stranded region during replication or transcription.

G4 at telomeres

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©P. Alberti

G4 at telomeres are considered challenging structures, in particular for the replication machinery and it is thought that helicases are needed to unfold these structures in cells. Our studies on contiguous G4, prompted us to study how single-stranded DNA binding proteins deal with telomeric contiguous G4. We focused our attention on the RPA (Replication Protein A) and POT1 (Protection of Telomere 1), two single-stranded DNA binding proteins acting at telomeres. The trimeric protein RPA is genome-wide; it is present at telomeres, during replication (and, likely, transcription); dysfunctional RPA leads to troubles in the synthesis of the G-strand. POT1, with its partner TPP1, is a subunit of the shelterin complex; it binds to TTAGGG repeats and, among its functions, it protects telomere ends from ATR-dependent DNA damage responses.

The figure here below synthetises our major results, detailed below.

Walking along telomeres: G4, hairpins and SSB proteins

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©P. Alberti

hRPA. After having characterised the how hRPA deal with a single telomeric G4 (doi: 10.1016/j.biochi.2014.04.006) and having highlighted a 5’ toward 3’ directionality in G4 unfolding   (doi: 10.1074/jbc.M115.709667), we have shown that RPA efficiently unfolds contiguous G4 formed by GGGTTA canonical telomeric repeats, despite their relatively high stability, independently of the number of contiguous G4 (doi: 10.1016/j.biochi.2017.11.017). This result has a biological relevance: it shows that, if contiguous G4s form during telomere replication or transcription, their number does not affect the efficiency of hRPA to coat the portion of the G-strand transiently exposed as a single-strand. This feature may be critical in ensuring telomere stability, by promptly suppressing or preventing the accumulation of contiguous G4 on the G-strand during telomere replication or transcription. It arises from the peculiar nature of telomeric G4, from their being independent and identical structural units, stable but not too stable. As a counterpoint, the binding efficiency of RPA to GGGTTA repeats strongly decreases in a particular non-physiological condition where the telomeric G4 units interact with each other (doi: 10.1093/nar/gkw003)

What happens when hairpins form in human telomeres. We next investigated an unstable variant telomeric motif. In the proximity of the subtelomeric regions of human telomeres, canonical GGGTTA repeats are interspersed with variant repeats (GGGTGA, GGGTCA, GGGGTT, …). The variant motif GGGCTA exhibits a length-dependent instability: sequences of more than ten GGGCTA repeats gain or loss repeats, likely through replication. We showed that GGGCTA repeats can form G4 and hairpins and that their hairpin form is poorly unfolded by RPA, suggesting a possible origin of GGGCTA repeats instability in telomeres  (doi: 10.1093/nar/gkab518). From the perspective of evolution, this study suggests that the instability of repeated sequences folding into hairpins may have led to a counterselection of hairpin-prone telomeric motifs, less efficiently unfolded by RPA. 

hPOT1-TPP1. We studied how the telomeric protein POT1 in complex with its partner TPP1 deals with contiguous telomeric G4. We showed that the structuring of the telomeric G-strand into contiguous G4 makes multiple POT1-TPP1 bind to it cooperatively and proceed from the 3’ end toward 5’ (doi: 10.1093/nar/gkab768). We did not observe this behaviour in lithium, where telomeric G4 are not stable. Thus, the structuring of the telomeric G-overhang into contiguous G4 affects the way in which multiple hPOT1-TPP1 bind to it.

Overall, our studies shed new light on telomeric G4s: as identical and independent structural units, stable but not overly stable, they are easily manageable structures for the proteins that have to unfold them. Telomeric G4s may indeed be telomeres’ best friend.