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Role of PARP1-interacting protein TRIM44 in the DNA damage response

DNA 손상 반응 과정에서 PARP1 결합 단백질인 TRIM44의 역할

  • 김용현 (KIM YONGHYEON, 아주대학교 일반대학원)

초록/요약

DNA double-strand breaks (DSBs) are most deleterious to cells and unrepaired DNA breaks cause cell death and genomic instability. To counteract this threat, cells have evolved the defense system to detect DNA damage, signal its presence and mediate its repair, so called DNA damage response (DDR). Two early major regulators of DDR and repair are poly(ADP-ribose) polymerase 1 (PARP1) and ataxia telangiectasia mutated (ATM) kinase. Although each function of these two proteins has been widely studied, their intercorrelations remain elusive. Here, I identified a novel PARP1-interacting protein TRIM44, a member of the tripartite motif (TRIM) family. I found that TRIM44 is recruited to DNA lesions in a PARP1 catalytic activity-dependent manner. I also found that the glutamic acid-rich (GR) domain of TRIM44 is essential for its interaction with PARP1. Functionally, TRIM44 protects the poly-ubiquitin chain of PARP1 via its zinc-finger ubiquitin binding (ZnF UBP) domain, thereby limiting PARP1 hyperactivation after DNA damage. Strikingly, TRIM44 deficiency inhibited the accumulation of ATM and MRN at damaged chromatin, thereby suppressing DSB repair. PARP inhibitors (PARPi) are known to inhibit the activity of PARP1 and promote PARP trapping at DNA lesions. PARPi hold promise for the treatment of tumors with homologous recombination (HR) repair defects, but PARPi resistance is still ubiquitous in the clinic. In a clonogenic survival assay, TRIM44 deficiency increased sensitivity to PARPi as efficiently as in BRCA1-deficient cells. Furthermore, PARPi resistance in 53BP1-deficient cells was overcome by TRIM44 depletion. Using data from The Cancer Genome Atlas (TCGA), I identified TRIM44 as an important target in renal clear cell carcinoma (KIRC) and found that sensitivity to DNA-damaging agent was increased in the absence of TRIM44. These data highlight that TRIM44 is an upstream regulator of PARP1 and ATM, and that inhibition of TRIM44 might be a way to significantly increase PARPi sensitivity in various cancers. Collectively, these data demonstrate that TRIM44 is recruited to DNA lesions in a PARP1-dependent manner and limits PARP1 hyperactivity by protecting PARP1 ubiquitination. This proper activation of PARP1 by TRIM44 promotes ATM-dependent DDR by inducing recruitment of the ATM and MRN complex to the site of DNA damage. In addition, the absence of TRIM44 increases sensitivity to DNA damaging agent and PARP inhibitor in several cancer cell lines.

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목차

I. INTRODUCTION 1
A. DNA damage response 1
B. Poly(ADP-ribose) polymerase 1 1
C. ATM-dependent DNA damage signaling pathway 4
D. Ubiquitination and deubiquitination 6
E. Tripartite motif proteins 8
F. PARP inhibitors in cancer therapy 8
G. Aim of this study 11
II. MATRIALS & METHODS. 12
1. Cell culture 12
2. Plasmid and RNA interference. 12
3. Laser micro irradiation and immunofluorescence 13
4. FokI assay 14
5. Antibodies and reagents 14
6. Western blotting 14
7. Pull-down assay 15
8. Whole-cell extraction 15
9. Immunoprecipitation 15
10. Nuclear fraction 15
11. Chromatin fractionation 16
12. Chromatin Immunoprecipitation (ChIP) 16
13. Alkaline Comet assay 17
14. Clonogenic survival assay 18
15. Statistical analysis 18
III. RESULTS. 19
1. Screening of TRIM family 19
2. TRIM44 is recruited at the DNA damage site(s) in PARP1-dependent manner 23
3. The Glu-rich domain of TRIM44 is required to interact with PARP1. 28
4. Absence of TRIM44 induces hyperactivation of PARP1 at DNA lesions 34
5. TRIM44 regulates the activation of PARP1 by protecting the poly-ubiquitin chain 40
6. Hyperactivation of PARP1 by the absence of TRIM44 prevents the binding of ATM and MRN complexes to DNA lesions 44
7. TRIM44 affects sensitivity to DNA damage reagent 48
8. TRIM44 affects sensitivity to PARP inhibitor 51
9. TRIM44 affects re-sensitivity to PARP inhibitor in PARP inhibitor-resistant cancer cells 55
10. TRIM44 has the potential to be a key gene in the treatment of clear cell renal cell carcinoma 58
IV. DISCUSSION 61
REFERENCES. 65
국문요약. 74

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