Elsevier

DNA Repair

Volume 12, Issue 7, July 2013, Pages 518-528
DNA Repair

The RECQL4 protein, deficient in Rothmund–Thomson syndrome is active on telomeric D-loops containing DNA metabolism blocking lesions

https://doi.org/10.1016/j.dnarep.2013.04.005Get rights and content

Highlights

  • RECQL4 preferentially unwinds telomeric substrates containing thymine glycol (Tg).

  • TRF2, a telomeric shelterin protein, stimulates RECQL4 helicase in presence of Tg.

  • RECQL4 binding is unchanged but TRF2 binds Tg-containing D-loops with less affinity.

  • WRN, but not RECQL4, stimulates NEIL1 glycosylase incision of the Tg lesion.

Abstract

Telomeres are critical for cell survival and functional integrity. Oxidative DNA damage induces telomeric instability and cellular senescence that are associated with normal aging and segmental premature aging disorders such as Werner Syndrome and Rothmund–Thomson Syndrome, caused by mutations in WRN and RECQL4 helicases respectively. Characterizing the metabolic roles of RECQL4 and WRN in telomere maintenance is crucial in understanding the pathogenesis of their associated disorders. We have previously shown that WRN and RECQL4 display a preference in vitro to unwind telomeric DNA substrates containing the oxidative lesion 8-oxoguanine. Here, we show that RECQL4 helicase has a preferential activity in vitro on telomeric substrates containing thymine glycol, a critical lesion that blocks DNA metabolism, and can be modestly stimulated further on a D-loop structure by TRF2, a telomeric shelterin protein. Unlike that reported for telomeric D-loops containing 8-oxoguanine, RECQL4 does not cooperate with WRN to unwind telomeric D-loops with thymine glycol, suggesting RECQL4 helicase is selective for the type of oxidative lesion. RECQL4's function at the telomere is not yet understood, and our findings suggest a novel role for RECQL4 in the repair of thymine glycol lesions to promote efficient telomeric maintenance.

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.

References (67)

  • T. Richter et al.

    Exp. Gerontol.

    (2007)
  • M.A. Rubio et al.

    Exp. Cell Res.

    (2004)
  • S.R. Werner et al.

    Biochem. Biophys. Res. Commun.

    (2006)
  • P. Aller et al.

    J. Mol. Biol.

    (2011)
  • R.C. Hayes et al.

    J. Mol. Biol.

    (1988)
  • S. Tornaletti et al.

    J. Biol. Chem.

    (2001)
  • K.-I. Takata et al.

    J. Biol. Chem.

    (2006)
  • T. von Zglinicki et al.

    Exp. Cell Res.

    (1995)
  • T. von Zglinicki

    Trends Biochem. Sci.

    (2002)
  • G.B. Morin

    Exp. Gerontol.

    (1997)
  • A.K. Ghosh et al.

    J. Biol. Chem.

    (2012)
  • A. Ghosh et al.

    J. Biol. Chem.

    (2009)
  • P.L. Opresko et al.

    Mol. Cell

    (2004)
  • M.L. Rossi et al.

    DNA Repair

    (2010)
  • A.N. Suhasini et al.

    J. Biol. Chem.

    (2009)
  • G.L. Dianov et al.

    J. Biol. Chem.

    (2000)
  • V. Bandaru et al.

    DNA Repair

    (2002)
  • A. Das et al.

    J. Biol. Chem.

    (2007)
  • V. Popuri et al.

    DNA Repair

    (2010)
  • L.L. Woo et al.

    Exp. Cell Res.

    (2006)
  • J.D. Griffith et al.

    Cell

    (1999)
  • S. Neidle et al.

    Curr. Opin. Struct. Biol.

    (2003)
  • P.L. Opresko et al.

    J. Biol. Chem.

    (2002)
  • J.Y. Kao et al.

    J. Biol. Chem.

    (1993)
  • S.D. Kathe et al.

    J. Biol. Chem.

    (2004)
  • B. Ahn et al.

    J. Biol. Chem.

    (2004)
  • L. Liu et al.

    Aging Cell

    (2002)
  • Z. Wang et al.

    PLoS Genet.

    (2010)
  • P. Hasty et al.

    Science

    (2003)
  • J.A. Harrigan et al.

    Nucleic Acids Res.

    (2006)
  • H. Ide et al.

    Nucleic Acids Res.

    (1985)
  • J.M. Clark et al.

    Biochemistry

    (1987)
  • R. Kusumoto et al.

    Biochemistry

    (2002)
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    Both these authors contributed equally to this study.

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