The RECQL4 protein, deficient in Rothmund–Thomson syndrome is active on telomeric D-loops containing DNA metabolism blocking lesions
Introduction
The five human RecQ helicases RECQL1, WRN, BLM, RECQL4 and RECQL5 are functionally significant in DNA maintenance, replication and DNA repair. In particular, WRN and RECQL4 have been shown to be important proteins for telomere maintenance [1], [2]. Telomeres protect human chromosome ends from degradation, and shortened and unstable telomeres are frequently associated with aging [3], [4], [5]. In accordance, cells from patients with segmental premature aging syndromes Werner syndrome (WS) and Rothmund–Thomson syndrome (RTS) deficient in WRN and RECQL4, respectively, exhibit telomere instability [2], [6]. While WRN has been extensively studied in regard to its role in DNA and telomere maintenance [6], [7], [8], [9], [10], [11], much less is known about the roles of RECQL4.
Telomeres can become unstable due to DNA damage, particularly that resulting from oxidative damage [2], [3], [4]. Oxidative DNA damage can be caused by external agents, for example by chemical oxidants and ionizing radiation, or endogenously, mainly by reactive oxygen species (ROS) formed by cellular aerobic metabolism [5]. Various oxidative DNA lesions exist such as: 6-diamino-4-hydroxy-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine (commonly known as Fapy G and Fapy A lesions, respectively), hydroxyl-methylamine and 8-oxoguanine which are commonly studied, and thymine glycol which is less well-characterized. In addition to exhibiting unstable telomeres, WS and RTS cells also show hypersensitivity to oxidative agents [6], [7], together suggesting a critical role for these RecQ helicases in the repair of oxidative lesions at the telomeres.
Generally, guanine is considered to be the most commonly oxidized base, due to its low oxidation potential. However, thymine glycol (Tg) is acknowledged as the most common oxidation product of the thymine base [8]. Because Tg base pairs efficiently with its normal Watson-Crick partner adenine, this lesion is only weakly mutagenic relative to other oxidative lesions [9]. Critically, however, Tg has been shown to be lethal to cells by strongly inhibiting DNA replication in vitro and in vivo in both E. coli [10], [11] and human cells [12]. Although human DNA translation polymerase eta (POL?) is reportedly capable of accurately replicating across Tg lesions in vitro [13], [14], it was also shown that traditional replicative and repair polymerases stall just one base beyond the Tg, inhibiting further replication and elongation [10], [11], [15]. Crystallography has demonstrated that replication arrest is actually caused by the local helical distortion created by base-pairing at a Tg lesion [8], [15], which also prevents efficient repair of these lesions at sites of double strand breaks [16]. Tg damage therefore presents a critical barrier to cell survival.
Supporting these observations is a multi-species comparative study which demonstrates that Tg damage in vivo is correlated to lifespan [17]. Interestingly, telomere length is also correlated with longevity in animals, human cells and individuals [18], [19], [20]. As WRN and RECQL4 are implicated strongly in telomere maintenance, and telomere length and integrity are disrupted by oxidative damage [21], [22], [23], one could speculate that their loss prevents efficient telomere maintenance around oxidative lesions such as Tg and leads to telomere instability, telomere loss and ultimately cellular senescence that are observed in WS and RTS patient cells under oxidative stress [7], [24], [25].
We recently reported that RECQL4 is involved in telomere maintenance in vivo, is functionally stimulated by a telomeric shelterin protein TRF2, and shows a preference to unwind a telomeric D-loop containing 8-oxoguanine lesions in vitro [26] similar to WRN [27]. To explore their possible role in the repair of replication-blocking Tg lesions in telomeres, we developed unique telomeric substrates containing Tg lesions and investigated in vitro the functional capacity of RECQL4 and WRN to unwind telomeric D-loops and replication forks containing Tg lesions. We show that unlike WRN, RECQL4 shows a clear preference to unwind substrates containing Tg and that this activity is stimulated on Tg D-loops in the presence of TRF2. We therefore propose that RECQL4 is functionally important for the cellular response to Tg lesions at the telomeres.
Section snippets
Preparation of oligonucleotides
Synthetic telomeric D-loops were constructed as described in Fig. 1 and Table 1. Unmodified oligonucleotides were manufactured and PAGE-purified by Integrated DNA Technologies (Coralville, IA, USA). All modified oligonucleotides containing thymine glycol were synthesized and PAGE-purified by The Midland Certified Reagent Company (Midland, TX, USA). Oligonucleotides were labeled, annealed and characterized as described previously [27]. Non-telomeric D-loop containing thymine glycol was also
RECQL4 shows a preference to unwind telomeric D-loops containing Tg
We and others have shown previously that BLM, WRN and RECQL4 have functional roles in telomere maintenance and can potentially unwind telomeric substrates in vitro. They also exhibit a preference for a telomeric structure containing an oxidized guanine base (8-oxoguanine) over an undamaged structure. While 8-oxoguanine is a mutational lesion, thymine glycol (Tg) is a replication-blocking lesion and therefore a highly critical lesion to repair for cellular survival. We investigated whether these
Discussion
Characterizing the metabolic roles of RECQL4 and WRN in telomere maintenance is crucial to understanding the pathogenesis of their associated disorders. The D-loop is a generally accepted telomeric structure that is formed to protect chromosome ends from degradation and is unwound during telomeric repair, replication and transcription [48], [49]. WRN, BLM and RECQL4 are all recognized to operate in telomeric maintenance and function in D-loop processing [26], [50], [51], [52], [53], [54], [55],
Conclusions
In the future, crystallographic analysis may further characterize the physical interactions with the Tg lesion and with TRF2 that are shown to stimulate RECQL4's helicase activity. Perhaps the helical distortion induced by Tg lesions is a more optimal conformation on which the RECQL4 helicase can specifically act; certainly this distortion is not optimal for TRF2 or TRF1 binding, which may result in unprotected and unstable telomeres. Structurally, RECQL4 is unique compared with WRN and the
Conflict of interest statement
None.
Acknowledgements
We thank Huiming Lu and Christopher Dunn, NIA, for critically reading the manuscript. We also thank Marie Rossi and Lale Dawut, NIA, for biochemical methods instruction and technical help, Christopher Dunn and Tomasz Kulikowicz, NIA, for purification of the RecQ helicase proteins, and Patricia Opresko, University of Pittsburgh, for purified shelterin proteins. This work was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.
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Both these authors contributed equally to this study.