Introduction
Tousled-like kinases (TLKs) are a class of Ser/Thr kinases implicated in critical cellular processes such as DNA replication and DNA damage repair in higher eukaryotes. Discovered through various studies, TLKs have been shown to play essential roles in maintaining genomic stability, particularly under conditions of replication stress and DNA damage. The TLK family comprises two homologs, TLK1 and TLK2, which share high sequence similarity, with 94% identity in the kinase domain and 84% overall sequence identity. Despite their similarities, TLK1 and TLK2 exhibit distinct biological roles, as evidenced by their differential involvement in developmental processes and DNA damage responses. This article explores the functions of TLK1, its interaction with various substrates, and its critical role in DNA damage repair mechanisms, particularly double-strand break (DSB) repair and homologous recombination repair (HRR).
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TLK1 and Its Role in DNA Damage Response
TLK1 has been extensively studied for its involvement in the DNA damage response (DDR) and replication processes. TLK1 depletion has been shown to delay S-phase progression, highlighting its crucial role in replication. One significant interaction involves TLK1 and human RAD9 during replication fork stalling. TLK1 phosphorylates RAD9 at Ser328, leading to the dissociation of the 9-1-1 complex and cytosolic localization of RAD9, which is crucial for deactivating the checkpoint post-DNA repair. Additionally, TLK1 exhibits chaperone functionality, recruiting RAD9 to the double-strand break (DSB) site, further emphasizing its role in DDR.
Another critical substrate of TLK1 is Asf1a/b, a histone chaperone phosphorylated by TLK1 during the S phase, facilitating the binding of the H3-H4 tetramer to newly replicated DNA. TLK1's interaction with chromatin, which decreases during replication stress, underscores its role in maintaining chromatin stability. Furthermore, a splice variant of TLK1, TLK1B, is expressed upon ionizing radiation (IR) exposure, providing radio resistance to cells and inducing UV resistance. This variant shares the conserved kinase domain with TLK1, indicating considerable substrate overlap and functional similarities.
Fig. 1 Mechanistic role of TLK1 in DNA damage repair (DDR). (Ghosh I., De Benedetti A. 2023)
TLK1 in DSB Repair
TLK1 interacts with numerous DNA damage and DSB repair proteins, such as NEK1, AKTIP, RAD54B, and FANCM. Recent studies have elucidated TLK1's specific role in regulating DSB repair via RAD54. RAD54, along with RAD51, is pivotal in the homologous recombination repair (HRR) process. TLK1 phosphorylates RAD54 at multiple sites, including T41, T59, and T700, affecting various stages of the HRR process.
Upon DSB induction by IR, TLK1 activity is transiently inhibited through a Chk1-dependent mechanism. TLK1 is phosphorylated by Chk1 at Ser695, marking its inactivity. This transient inhibition is followed by a recovery phase where TLK1 re-phosphorylates its substrates, including RAD54. This dynamic phosphorylation-dephosphorylation cycle is crucial for the proper functioning of RAD54 in HRR, facilitating the repair process. TLK1's interaction with RAD54 also suggests a regulatory mechanism where TLK1 ensures the timely relocalization of RAD54 and RAD51 from the cytoplasm to the nucleus during DSB repair.
Role of TLK1 in HRR Factors Regulation
TLK1's phosphorylation of RAD54 regulates different stages of HRR, highlighting its role in modulating HRR dynamics. Phosphorylation at RAD54's N-terminal domain affects its interaction with HRR proteins like RAD51AP1, NUCKS1, and CDK2, while phosphorylation at the C-terminal domain influences its interaction with double-strand DNA templates. These phosphorylation events by TLK1 are critical for maintaining the balance between replication fork progression and regression under stress conditions.
Moreover, TLK1's involvement in chromatin assembly during replication and its interaction with histone chaperone Asf1 suggest complex regulatory functions beyond mere chromatin remodeling. TLK1's role in the phosphorylation of NEK1, a co-activator of ATR, further underscores its importance in the intra-S-phase checkpoint and replication stress response.
TLK1 in Eukaryotic Recombination Repair
TLK1's activity is vital for homologous recombination repair (HRR). Activation of TLK1 increases HRR activity, while its depletion significantly decreases HRR efficiency. TLK1's interaction with RIF1, a key player in the decision-making process between NHEJ and HRR, suggests a role in regulating the choice of repair pathway. TLK1 also interacts with RAD54B, another RAD54 paralog, implicating its involvement in chromosome segregation and possibly telomeric recombination.
TLK1's preferential localization to the nucleolus, even without DNA damage induction, indicates potential roles in ribosomal biogenesis and DSB repair within this highly compacted chromatin environment. The interplay between TLK1 and various substrates underscores its multifaceted role in maintaining genomic stability.
TLKs and DNA Damage and Checkpoint Functions
Beyond their roles in DSB repair, TLKs are involved in the repair of other types of DNA lesions, such as UV-induced thymidine dimers and cisplatin-induced interstrand crosslinks (ICLs). Studies have shown that TLK2, in particular, modulates the DNA damage response (DDR) and G2 recovery by regulating Asf1A, a histone chaperone involved in chromatin assembly. TLK1B, a splice variant of TLK1, also plays a protective role against UV radiation, enhancing chromatin assembly and repair efficiency.
TLK1's overexpression in normal cells facilitates the repair of UV-induced DNA damage, suggesting a broader role in chromatin remodeling and possibly direct involvement in the repair process. These findings highlight the importance of TLKs in various DNA repair pathways and their potential as therapeutic targets in cancer treatment.
Conclusion
Tousled-like kinases (TLKs), particularly TLK1, play critical roles in DNA damage repair and replication stress response. Through interactions with various substrates like RAD9, RAD54, and Asf1, TLK1 orchestrates complex regulatory mechanisms that ensure genomic stability. Its involvement in DSB repair, HRR, and checkpoint functions underscores its multifaceted role in maintaining cellular homeostasis under stress conditions. Understanding the precise functions of TLKs and their regulatory mechanisms provides valuable insights into potential therapeutic strategies for enhancing DNA repair and combating genomic instability-related diseases.
References
- Groth A., et al. Human Tousled like kinases are targeted by an ATM‐and Chk1‐dependent DNA damage checkpoint. The EMBO Journal. 2003, 22(7):1676-87.
- Ghosh I., De Benedetti A. Untousling the role of tousled-like kinase 1 in DNA damage repair. International Journal of Molecular Sciences. 2023, 24 (17): 13369.
- Ehsan H. Molecular basis and functional diversity of TOUSLED kinase. Bioresearch Communications-(BRC). 2020, 6 (1): 840-3.
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