We show that telomere-specific proteins, POT1, TRF1 and TRF2 stimulate the binding and enzymatic activities of the LP-BER proteins APE1, FEN1 and LigI both individually and when they act together in reconstituted LP-BER using a telomeric substrate. Because telomeres contain a high concentration of guanine repeat sequences, they are more susceptible to oxidative damage than the rest of the genome. The oxidative damage product 8-oxo-guanine (8-oxo-G) is the most abundant base lesion detected at the telomeres,61
indicating that BER must be a frequently required repair pathway. Previous work showed that oxidative damage causes disruption of TRF1 and TRF2 binding, leading to depletion of the shelterin components from the telomeres.62
In addition to maintaining the optimal concentration of the shelterin components at the telomeres, the benefit of minimizing oxidative base damage in telomeres is the prevention of early cell senescence or apoptosis caused by telomere dysfunction. Therefore, we propose that telomere-specific proteins POT1, TRF1 and TRF2 have evolved to enhance BER at telomeres.
Muftuoglu et al. found that pol β and FEN1 both interacted with the telomere-specific protein TRF2. Association with pol β is further supported by the previous observation that TRF2 colocalized with the polymerase outside of the telomeric regions.60
This suggests a physical connection between telomere- and BER-specific proteins. We further asked whether the other telomeric DNA binding proteins interact with the BER pathway, demonstrating that POT1 binds both FEN1 and pol β, and TRF1 binds FEN1. Considering that all three telomeric proteins bind FEN1, it is reasonable to assume that they aid the nuclease in accessing telomeric DNA to find flap intermediates of LP-BER. This concept is consistent with structural evidence for coordinated protein function in BER found by Parikh et al.63
and Mol et al.64
Their results suggest that BER works through a systematic recognition of enzyme complexes formed at each step of LP-BER, presumably diminishing the probability that intermediates could generate further DNA damaging events. Wilson and Kunkel65
referred to this stepwise mechanism as “passing the baton” from APE1 cleavage to pol β lyase and DNA synthesis to ligation during SP-BER. Prasad et al.,66
however, showed that if FEN1 is involved, as in LP-BER, it must be recruited to the enzymatic complex. Our results suggest that one mechanism is by binding shelterin proteins.
These results contrast with analyses of repair protein accessibility to DNA in reconstituted chromatin, where the nucleosome acts as a barrier to the repair proteins, unlike the shelterin components. DNA glycosylase, pol β, FEN1 and LigI were all less efficient catalytically when tested on a reconstituted mononucleosome.67
Moreover, the extent of inhibition of activity depended on both the position of the substrate structure along the histone core and the orientation of the helix with respect to the histones. This behavior is consistent with physical occlusion of the substrate site requiring enzyme activity. Since chromatin hosts BER, additional mechanisms must partially or completely reverse the occlusion during the repair process. Our reconstitutions with telomere proteins show no evidence of occlusion. Moreover, the protein-coated damaged DNA is not simply invisible to the repair complex. Instead, the binding and stimulation properties of the telomeric proteins suggest that BER complexes are specifically recruited to telomeres, and that their repair functions are promoted. The evolution of these mechanisms suggests that efficient BER in telomeres is important for long-term species survival.
All three shelterin proteins stimulated the nuclease activity of FEN1. A more refined view of the results is informative. POT1 exhibited greater stimulation on the 2-nt telomeric flap compared with the non-telomeric analog, suggesting that POT1 has transient binding capacity to this structure, and that binding improves stimulation (). However, binding of POT1 to a 13-nt flap was less stimulatory to FEN1, suggesting that the system has evolved to work on short flaps. In fact, comparing overall stimulation from 2-nt flap cleavage to 13-nt flap cleavage, FEN1 was promoted most by shelterin proteins when the flap was short, implying that the system disfavors formation and processing of long flaps. This conclusion is further supported by the observation that Dna2 nuclease/helicase, which can activate replication protein A-coated flaps for cleavage by FEN1 in Okazaki fragment processing, is ineffective with POT1-coated flaps (unpublished observations). Because telomeric sequences are highly repetitive, they could be subject to sequence disruptions from polymerase slippage by pol β.68
Avoidance of long-flap intermediates may suppress this effect. TRF1 and TRF2 stimulation of FEN1 is also enhanced on a telomeric substrate compared with a nontelomeric substrate. TRF1 and TRF2 interact with and bend the telomeric dsDNA,68–70
a possible mechanism to make the flap base more accessible to FEN1.
We observed moderate stimulation of APE1 cleavage and LigI joining by POT1, TRF1 and TRF2, even though we could detect no physical interaction with the telomere proteins. This resembles the action of APE1, which improves OGG1 glycosylase activity without apparent physical interaction.71–73
POT1 indirectly interacts with dsDNA within the shelterin complex (reviewed in ref. 42
). We speculate that POT1 transiently interacts with APE1 and LigI, facilitating a long-lived structural conformation change that leads to an increase in the biological efficiency of BER. Native gel electrophoresis experiments indicate that POT1 stabilizes the interaction of APE1 and LigI for their respective substrates (Figs. S2 and S6
APE1 binding to an abasic site is critical for prevention of subsequent damage, leading to abortive intermediates.21
Further oxidation or reduction of the 5-dRP moiety formed by APE1 or damage induction not only inhibits lyase activity of pol β but also forms DNA/protein cross-links (DPC) with pol β.21,32,35,74,75
Once bound, APE1 kinks and surrounds the AP strand and cleaves the sugar-phosphate backbone. APE1 then facilitates loading of pol β. POT1 may only weakly interact with APE1, so that it does not disrupt the complex interaction APE1 must have with its substrate. Because POT1 physically interacts with pol β, it may help APE1 direct pol β to its substrate. Similarly, POT1 may have evolved to avoid interference with the LigI mechanism, which involves encircling the nick site.
TRF1 and TRF2 stimulate APE1 and LigI preferentially on telomeric substrates (, S1B, 4B and S5B
). Although TRF1 and TRF2 lose binding capacity if a guanine is converted to an abasic site,62
they may act from an adjacent site. Alternatively, shelterin components may recruit APE1 and LigI indirectly, by exposing damage sites and allowing access for the repair enzymes.
While the shelterin proteins stimulate BER on both telomeric and non-telomeric substrates, the localization of shelterin to telomeres is consistent with the conclusion that it exerts its effects on DNA repair in the telomere environment. Our results show stimulation of LP-BER. In stark contrast, Rochette and Brash50
showed that the inhibition of NER in telomeres led to higher incidence of cyclobutane pyrimidine dimers. Furthermore, TRF2 inhibits the XPF-ERCC1 endonuclease in vivo, which is required for NER.76,77
The types of damage NER repairs are recognized by distortion of helical structure caused by the presence of bulky adducts or unnatural covalent bonds. TRF2 also interacts with and inhibits the ATM kinase to suppress DNA double-strand break repair at ends of telomeres.78
Furthermore, Fink et al. discovered that targeting of a key NHEJ protein, Ku80, in the absence of apparent DNA damage, contributes to telomere dysfunction through TRF2 abstraction from the telomere.79
A reasonable set of conclusions is that base damage is a very prevalent lesion in telomeres and NER substrates, and double-strand breaks are less common.
Although telomeres are non-coding DNA, their integrity is vital to cell health. However, their triplet guanine-containing repeat sequence makes them particularly susceptible to dangerous shortening after oxidative damage. Our results indicate that they have evolved a way to promote efficient repair of this damage by recruitment of LP-BER proteins.