Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities.

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response.

Nucleostemin deletion reveals an essential mechanism that maintains the genomic stability of stem and progenitor cells.

Stem and progenitor cells maintain a robust DNA replication program during the tissue expansion phase of embryogenesis. The unique mechanism that protects them from the increased risk of replication-induced DNA damage, and hence permits self-renewal, remains unclear. To determine whether the genome integrity of stem/progenitor cells is safeguarded by mechanisms involving molecules beyond the core DNA repair machinery, we created a nucleostemin (a stem and cancer cell-enriched protein) conditional-null allele and showed that neural-specific knockout of nucleostemin predisposes embryos to spontaneous DNA damage that leads to severe brain defects in vivo. In cultured neural stem cells, depletion of nucleostemin triggers replication-dependent DNA damage and perturbs self-renewal, whereas overexpression of nucleostemin shows a protective effect against hydroxyurea-induced DNA damage. Mechanistic studies performed in mouse embryonic fibroblast cells showed that loss of nucleostemin triggers DNA damage and growth arrest independently of the p53 status or rRNA synthesis. Instead, nucleostemin is directly recruited to DNA damage sites and regulates the recruitment of the core repair protein, RAD51, to hydroxyurea-induced foci. This work establishes the primary function of nucleostemin in maintaining the genomic stability of actively dividing stem/progenitor cells by promoting the recruitment of RAD51 to stalled replication-induced DNA damage foci.

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

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.

Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1.

Continuously dividing cells must be protected from telomeric and nontelomeric DNA damage in order to maintain their proliferative potential. Here, we report a novel telomere-protecting mechanism regulated by nucleostemin (NS). NS depletion increased the number of telomere damage foci in both telomerase-active (TA(+)) and alternative lengthening of telomere (ALT) cells and decreased the percentage of damaged telomeres associated with ALT-associated PML bodies (APB) and the number of APB in ALT cells. Mechanistically, NS could promote the recruitment of PML-IV to SUMOylated TRF1 in TA(+) and ALT cells. This event was stimulated by DNA damage. Supporting the importance of NS and PML-IV in telomere protection, we demonstrate that loss of NS or PML-IV increased the frequency of telomere damage and aberration, reduced telomeric length, and perturbed the TRF2(ΔBΔM)-induced telomeric recruitment of RAD51. Conversely, overexpression of either NS or PML-IV protected ALT and TA(+) cells from telomere damage. This work reveals a novel mechanism in telomere protection.

Nucleostemin inhibits TRF1 dimerization and shortens its dynamic association with the telomere.

TRF1 is a key component of the telomere-capping complex and binds double-strand telomeric DNA as homodimers. So far, it is not clear whether TRF1 dimerization coincides with its telomere binding or is actively controlled before it binds the telomere, and in the latter case, how this event might affect its telomere association. We previously found that TRF1 dimerization and its telomere binding can be increased by GNL3L, which is the vertebrate paralogue of nucleostemin (NS). Here, we show that NS and GNL3L bind TRF1 directly but competitively through two separate domains of TRF1. In contrast to GNL3L, NS prevents TRF1 dimerization through a mechanism not determined by its ability to displace TRF1-bound GNL3L. Furthermore, NS is capable of shortening the dynamic association of TRF1 with the telomere in normal and TRF2(ΔBΔM)-induced telomere-damaged cells without affecting the amount of telomere-bound TRF1 proteins in vivo. Importantly, NS displays a protective function against the formation of telomere-dysfunction-induced foci. This work demonstrates that TRF1 dimerization is actively and oppositely regulated by NS and GNL3L extrachromosomally. Changing the relative amount of TRF1 monomers versus dimers in the nucleoplasm might affect the dynamic association of TRF1 with the telomere and the repair of damaged telomeres.

GNL3L stabilizes the TRF1 complex and promotes mitotic transition.

Telomeric repeat binding factor 1 (TRF1) is a component of the multiprotein complex "shelterin," which organizes the telomere into a high-order structure. TRF1 knockout embryos suffer from severe growth defects without apparent telomere dysfunction, suggesting an obligatory role for TRF1 in cell cycle control. To date, the mechanism regulating the mitotic increase in TRF1 protein expression and its function in mitosis remains unclear. Here, we identify guanine nucleotide-binding protein-like 3 (GNL3L), a GTP-binding protein most similar to nucleostemin, as a novel TRF1-interacting protein in vivo. GNL3L binds TRF1 in the nucleoplasm and is capable of promoting the homodimerization and telomeric association of TRF1, preventing promyelocytic leukemia body recruitment of telomere-bound TRF1, and stabilizing TRF1 protein by inhibiting its ubiquitylation and binding to FBX4, an E3 ubiquitin ligase for TRF1. Most importantly, the TRF1 protein-stabilizing activity of GNL3L mediates the mitotic increase of TRF1 protein and promotes the metaphase-to-anaphase transition. This work reveals novel aspects of TRF1 modulation by GNL3L.

Histopathology of corneal melting associated with diclofenac use after refractive surgery.

PURPOSE: To describe the histopathology of the cornea in 3 cases of corneal melting associated with diclofenac therapy after refractive surgery procedures. SETTING: Clinic and pathology laboratory. METHODS: Three cases of corneal melting associated with diclofenac therapy (2 after laser in situ keratomileusis [LASIK] and 1 after mini-radial keratectomy enhancement of a LASIK undercorrection) were studied using patient and referring physician interviews, chart reviews, and histopathologic examination of the corneal tissue. RESULTS: In all 3 cases, the flaps were dislocated and the stromal corneal bed was exposed. Diclofenac, generic or brand name, was used in all cases; in 1 case, both generic and brand name were used. Dosing and duration varied, but in all 3 cases diclofenac was used at least 4 times a day for at least 3 days after LASIK. Topical steroids were also prescribed, but 1 patient did not use them. Preoperative medical conditions were present in 2 cases. Histologic analysis showed evidence of an inflammatory response in advanced cases and keratolysis and lack of inflammatory cells in the flaps that were amputated early. CONCLUSIONS: The use of generic or brand-name diclofenac with or without adjunctive topical steroids after LASIK can be associated with corneal melting when the LASIK flap is dislodged and the corneal stromal bed exposed. Caution is recommended with diclofenac use after LASIK in such cases.