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Phosphorylation enhances Asf1 binding to soluble cellular histones. (a) Western blot analysis of e-Asf1a complexes isolated from conditional cell lines expressing e-Asf1a wt/4D/4A. Cytosolic complexes were obtained from cells released into mid-S phase from a thymidine block. (b) Gel-filtration analysis of cytosolic extracts from S phase cells conditionally expressing e-Asf1a wt/4D/4A. Similar induction of e-Asf1a wt/4D/4A was verified (Supplementary a). Two distinct Asf1 complexes are indicated. (c) In vitro competition assay. Whole-cell extracts of HeLa S3 cells expressing H3.1-HA-FLAG20 were pre-incubated with mixture of recombinant Asf1a wt and phospho-mimetic (4E) mutant or with Asf1a V94R. e-H3.1 complexes were purified and binding of recombinant Asf1a assessed taking advantage of the difference in mobility between wt and 4E. The ratio of Asf1a 4E relative to wt in the bound and unbound fractions was quantified in three independent experiments as depicted in the graph. The mean with s.d is shown, **P=0.0063 (unpaired t-test).
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During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation has an impact on human...
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... level of phosphorylation decreased gradually upon shortening of the C-terminal tail of Asf1a, and removal of the entire tail reduced phosphorylation by 85% compared to the wild-type (Fig. 1c, compare 1-156 and wt). Single serine to alanine substitutions as well as simultaneous mutation of the sites identified in vivo (Asf1a pS166, pS175) had a moderate effect (Fig. 1c, see Asf1a 2A; Supplementary Fig. 4a). However, mutation of all four sites identified in vitro reduced phosphorylation to the same extent as removing the full Asf1a tail (Fig. 1c, compare Asf1a 4A and 1-156). ...
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... therefore tested the stability of our Asf1a phospho-mimetic (4D) and phosphorylation deficient (4A) mutants in cycloheximide (CHX) treated cells. We did not observe significant differences in stability ( Supplementary Fig. 4a). However, we noted that CHX treatment strongly stimulated Asf1 phosphorylation ( Supplementary Fig. 4b, Fig. 6b), which due to the mobility shift might influence stability measurements of the wild-type protein. ...
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... did not observe significant differences in stability ( Supplementary Fig. 4a). However, we noted that CHX treatment strongly stimulated Asf1 phosphorylation ( Supplementary Fig. 4b, Fig. 6b), which due to the mobility shift might influence stability measurements of the wild-type protein. ...
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... of either Asf1a wild-type or the phospho-mutants did not significantly perturb the cell cycle ( Supplementary Fig. 4c). We thus asked whether the mutants could substitute for endogenous Asf1a and rescue cells arrested in S phase due to co-depletion of Asf1a and Asf1b (Fig. 3). ...
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... used our conditional cell lines to purify complexes from cells synchronized in mid S phase. Comparison of cytosolic complexes, representing highly soluble cytoplasmic and nuclear Asf1 11 , showed that the phospho-mimetic Asf1a mutant binds more histone H3, new histone H4 acetylated at lysine 12, Importin-4, NASP and RbAp46/48 than wild-type Asf1a (Fig. 4a). Importin-4, NASP and RbAp46/48 form a complex with Asf1 through mutual binding to histone H3- H4 11 , and their abundances are thus expected to mirror histone binding as we observe. Previous work has shown that Asf1 distributes into a histone-bound multi-chaperone complex and a histone-free form in cytosolic extracts from S phase ...
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... work has shown that Asf1 distributes into a histone-bound multi-chaperone complex and a histone-free form in cytosolic extracts from S phase cells 6 . Analysis of the phospho-mutants by gelfiltration demonstrated a clear shift of the Asf1a phospho-mimetic mutant (4D) towards the histone-bound complex relative to the wild-type and 4A proteins ( Fig. 4b, Supplementary Fig. 5a). Consistent with this, S166 phosphorylation on endogenous Asf1a could also be detected in the histone-bound complex ( Supplementary Fig. 5b). Moreover, TLK1 phosphorylation of GST-Asf1a enhanced histone H3-H4 binding in cell extracts ( Supplementary Fig. 5c). These data imply that phosphorylated Asf1 might bind soluble cellular ...
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... and phospho-mimetic proteins to histones in a cell extract. Recombinant Asf1a wild-type and phospho-mimetic mutant (4E) were added together to an extract of HeLa S3 cells expressing H3.1-HA-FLAG (e-H3.1). Upon isolation of e-H3.1 complexes, we found a significant enrichment of the phospho-mimetic mutant relative to recombinant wild-type Asf1a (Fig. 4c). Importantly, this was paralleled by a larger depletion of the phospho-mimetic mutant from the unbound fraction. Asf1a containing a mutated histone-binding pocket (V94R) 25 did not bind histones in the ...
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... to wt in the bound and unbound fractions was quantified in three independent experiments as depicted in the graph. The mean with s.d is shown, **p=0.0063 (unpaired t test). (a) Western blot analysis of e-Asf1a complexes isolated from conditional cell lines expressing e-Asf1a wt/4D/4A. Nuclear extracts were prepared from cells synchronized as in Fig. 4a. (b) SILAC-based proteomic analysis of e-Asf1a 4D and wt complexes. Complexes were isolated from whole extracts of conditional cells grown in heavy (e-Asf1a 4D) and light (e-Asf1a wt) amino acids, mixed in a 1:1 ratio and analyzed by mass spectrometry. The relative enrichment in e-Asf1a 4D versus wt complexes of known binding partners ...
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Histone H1.0 belongs to the class of linker histones (H1), although it is substantially distinct from other histone H1 family members. The differences can be observed in the chromosomal location and organization of the histone H1.0 encoding gene, as well as in the length and composition of its amino acid chain. Whereas somatic (H1.1–H1.5) histone H...
Fluctuations in the ambient environment can trigger chromatin disruptions, involving replacement of nucleosomes or exchange of their histone subunits. Unlike canonical histones, which are available only during S-phase, replication-independent histone variants are present throughout the cell cycle and are adapted for chromatin repair. The H2A.Z vari...
Citations
... The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are conserved serine-threonine kinases that function in numerous cellular processes, including DNA replication, DNA repair, transcription, and chromatin maintenance. 1 Both TLK1 and TLK2 interact with and regulate the ASF1A and ASF1B histone H3-H4 chaperones. [2][3][4][5][6][7] Depletion of both TLK1 and TLK2, or both ASF1A and ASF1B, led to overlapping cellular phenotypes, including DNA damage, innate immune activation and the induction of the alternative lengthening of telomeres (ALT) pathway. [8][9][10][11] TLK1 and TLK2 are regulated by the DNA damage response and the phosphorylation of TLK1 on its C-terminus by the checkpoint kinase CHK1 inhibits TLK1 activity. ...
... We next examined the phosphorylation of two reported TLK1 substrates ASF1A and NEK1. 6,37 In both cases, no significant difference in the phosphorylation of either substrate could be observed between the cell lines ( Figures 2E and 2F). ...
The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants underlie the neurodevelopmental disorder (NDD) ‘Intellectual Disability, Autosomal Dominant 57’ (MRD57), characterized by intellectual disability and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a heterozygous TLK1 variant (c.1435C>G, p.Q479E), as well as a mutation in MDM1 (c.1197dupT, p.K400∗). Cells expressing TLK1 p.Q479E exhibited reduced cytokine responses and elevated DNA damage, but not increased radiation sensitivity or DNA repair defects. The TLK1 p.Q479E variant impaired kinase activity but not proximal protein interactions. Our study provides the first functional characterization of NDD-associated TLK1 variants and suggests that, such as TLK2, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.
... Tousled-like kinases (TLKs) are pivotal for genome stability and normal development across species (Mortuza et al., 2018), with peak activity during the S-phase (Ehsan et al., 2004). They're also targeted by DNA damage checkpoints (Groth et al., 2003) and impact antisilencing factor 1 (ASF1), influencing chromatin structure (Silljé and Nigg, 2001;Klimovskaia et al., 2014). TLK2 mutations have been linked to neurological disorders, such as mental disabilities, behavior disorders, autism spectrum disorder (Lelieveld et al., 2016;Reijnders et al., 2018), and Alzheimer's disease (Wang et al., 2010). ...
Background: Tousled-like kinase 2 (TLK2) is integral to DNA repair, replication, and cell cycle regulation, crucial for maintaining genome stability and integrity. However, the expression and prognostic value of TLK2 in hepatitis B viral (HBV) -related hepatocellular carcinoma (HCC) remains unclear.
Methods: We examined TLK2 expression and prognostic implications in pan-cancer by using diverse databases. Subsequently, TLK2 expression in HBV-related HCC tissues and adjacent tissues was assessed using quantitative real-time PCR and immunohistochemistry. The prognostic value of TLK2 was assessed through ROC curves, time-dependent ROC curves, Cox regression, Kaplan-Meier curve, and decision curve analysis. Additionally, analyses of immune infiltration, protein-protein interactions, key molecules of tumor-related signaling pathways, molecular subtypes, and TLK2-associated differentially expressed genes (DEGs) were conducted, along with GO/KEGG and GSEA enrichment analyses.
Results: TLK2 expression was significantly higher in HCC tissues compared to adjacent tissues and correlated with gender, AFP levels, albumin-bilirubin (ALBI) grade, microvascular invasion (MVI), maximum tumor diameter, tumor number, and TNM stage. TLK2 overexpression emerged as an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in HBV-related HCC patients. An integrated OS nomogram model, incorporating TLK2, age, ALBI grade, MVI, and tumor number, displayed enhanced prognostic capability (C-index: 0.765, 95% CI: 0.732–0.798) in predicting OS and has a higher net benefit than the TNM stage. Moreover, TLK2 expression correlated closely with immune cell infiltration and key molecules of signaling pathways. Functional enrichment analyses highlighted significant associations with DNA duplex unwinding, double-strand break repair, DNA replication, cell cycle, E2F targets, G2M checkpoint, and MYC targets V1.
Conclusion: TLK2 is notably overexpressed in HBV-related HCC and emerges as a promising prognostic biomarker, necessitating further validation.
... TLK1 can directly bind to chromatin and its interaction with chromatin has been also linked to replication stress. During replication stress TLK1 binding to chromatin decreases [8,9] TLK1B, a splice variant of TLK1 is expressed upon ionizing radiation (IR) exposure and provides radio-resistance to cells. TLK1B overexpression has been also shown to induce UV resistance [10]. ...
DNA damage repair lies at the core of all cells’ survival strategy, including cancerous. Therefore, targeting such repair mechanisms forms the major goal of cancer therapeutics. The mechanism of DNA repair has been tousled with the discovery of multiple kinases. Recent studies on Tousled like Kinases have brought significant clarity on the effectors of these kinases which stands to regulate DSB repair. In addition to their well-established role in the DDR and cell cycle checkpoint mediation after DNA damage or inhibitors of replication, their suspected involvement in the actual DSB repair process has more recently been strengthened by the important finding that TLK1 phosphorylates RAD54 and regulates some of its activities and localization in the cell. Earlier findings of its regulation of RAD9 during checkpoint deactivation as well as defined steps during NHEJ ends processing were earlier hints of its important involvement broadly in DSB repair. All this has opened up new avenues to target cancer cells in combination therapy with genotoxins and TLK inhibitors.
... Another downstream target of TLK1 is Asf1a/b which is phosphorylated at its C-terminus during S-phase and that leads to binding of H3-H4 tetramer that is assembled on newly replicated DNA. TLK1 can directly bind to chromatin and its interaction with chromatin has been also linked to replication stress, upon which, TLK1 binding to chromatin decreases [7,8]. TLK1B, a splice variant of TLK1 is expressed upon IR exposure and provides radio-resistance to cells. ...
... repaired DNA strands [8]. In another model organism T. brucei, TLK1 interacts with Asf1a and Asf1b to phosphorylate the histone chaperones which helps to maintain their activity [5]. ...
DNA damage repair lies at the core of all cells’ survival strategy, including cancerous. Therefore, targeting such repair mechanisms forms the major goal of cancer therapeutics. The mechanism of DNA repair has been tousled with the discovery of multiple kinases. Recent studies on Tousled like Kinases have brought significant clarity on the effectors of these kinases which stands to regulate DSB repair. In addition to their well-established role in the DDR and cell cycle checkpoint mediation after DNA damage or inhibitors of replication, their suspected involvement in the actual DSB repair process has more recently been strengthened by the important finding that TLK1 phosphorylates RAD54 and regulates some of its activities and localization in the cell. Earlier findings of its regulation of RAD9 during checkpoint deactivation as well as defined steps during NHEJ ends processing were earlier hints of its important involvement broadly in DSB repair. All this has opened up new avenues to target cancer cells in combination therapy with genotoxins and TLK inhibitors.
... This phosphorylation event promotes proper chromosomal condensation during metaphase and provides radioresistance in healthy mouse breast epithelial cells (MM3MG) and yeast [70]. TLK1B also phosphorylates the histone H3-H4 chaperones Asf1a and Asf1b, enhancing their affinity to bind to H3-H4 and enabling nucleosomal assembly during replication and damage repair [71,72]. Moreover, TLK1B phosphorylates Rad9, a component of the Rad9-Rad1-Hus1 (9-1-1) heterotrimeric complex involved in DNA repair [73]. ...
... TLKs play vital roles in various essential cellular processes, including chromatin formation, DNA replication, repair, and transcription [63,71,[122][123][124]. Studies conducted on mice and cells indicate that TLK1 and TLK2 have similar functions in developing and maintaining the genome's integrity [125]. ...
Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer (PCa) treatment, with success in developing more effective inhibitors of androgen synthesis and antiandrogens in clinical practice. However, hormone deprivation and AR ablation have caused an increase in ADT-insensitive PCas associated with a poor prognosis. Resistance to ADT arises through various mechanisms, and most castration-resistant PCas still rely on the androgen axis, while others become truly androgen receptor (AR)-independent. Our research identified the human tousled-like kinase 1 (TLK1) as a crucial early mediator of PCa cell adaptation to ADT, promoting androgen-independent growth, inhibiting apoptosis, and facilitating cell motility and metastasis. Although explicit, the growing role of TLK1 biology in PCa has remained underrepresented and elusive. In this review, we aim to highlight the diverse functions of TLK1 in PCa, shed light on the molecular mechanisms underlying the transition from androgen-sensitive (AS) to an androgen-insensitive (AI) disease mediated by TLK1, and explore potential strategies to counteract this process. Targeting TLK1 and its associated signaling could prevent PCa progression to the incurable metastatic castration-resistant PCa (mCRPC) stage and provide a promising approach to treating PCa.
... yeast [70]. TLK1B also phosphorylates the histone H3-H4 chaperones Asf1a and Asf1b, enhancing their affinity to bind to H3-H4 and enabling nucleosomal assembly during replication and damage repair [71,72]. Moreover, TLK1B phosphorylates Rad9, a component of the Rad9-Rad1-Hus1 (9-1-1) heterotrimeric complex involved in DNA repair [73]. ...
... TLKs play vital roles in various essential cellular processes, including chromatin formation, DNA replication, repair, and transcription [63,71,[124][125][126]. Studies conducted on mice and cells indicate that TLK1 and TLK2 have similar functions in developing and maintaining the genome's integrity [127]. ...
Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer (PCa) treatment, with success in developing more effective inhibitors of androgen synthesis and antiandrogens in clinical practice. However, hormone deprivation and AR ablation have caused an increase in ADT-insensitive PCas’ associated with poor prognosis. Resistance to ADT arises through various mechanisms, and most castration-resistant PCas’ still rely on the androgen axis, while others become truly androgen receptor (AR)-independent. Our research identified the human tousled-like kinase 1 (TLK1) as a crucial early mediator of PCa cell adaptation to ADT, promoting androgen-independent growth, inhibiting apoptosis, and facilitating cell motility and metastasis. Although explicit, the growing role of TLK1 biology in PCa has remained under-represented and elusive. In this review, we aim to highlight the diverse functions of TLK1 in PCa, shed light on the molecular mechanisms underlying the transition from androgen-sensitive (AS) to an androgen-insensitive (AI) disease mediated by TLK1, and explore potential strategies to counteract this process. Targeting TLK1 and its associated signaling could prevent PCa progression to the incurable metastatic castration-resistant PCa (mCRPC) stage and provide a promising approach to treating PCa.
... This phosphorylation event promotes proper chromosomal condensation during metaphase and provides radioresistance in healthy mouse breast epithelial cells (MM3MG) and yeast [70]. TLK1B also phosphorylates the histone H3-H4 chaperones Asf1a and Asf1b, enhancing their affinity to bind to H3-H4 and enabling nucleosomal assembly during replication and damage repair [71,72]. Moreover, TLK1B phosphorylates Rad9, a component of the Rad9-Rad1-Hus1 (9-1-1) heterotrimeric complex involved in DNA repair [73]. ...
... TLKs play vital roles in various essential cellular processes, including chromatin formation, DNA replication, repair, and transcription [63,71,[124][125][126]. Studies conducted on mice and cells indicate that TLK1 and TLK2 have similar functions in developing and maintaining the genome's integrity [127]. ...
Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer (PCa) treatment, with success in developing more effective inhibitors of androgen synthesis and antiandrogens in clinical practice. However, hormone deprivation and AR ablation have caused an increase in ADT-insensitive PCas’ associated with poor prognosis. Resistance to ADT arises through various mechanisms, and most castration-resistant PCas’ still rely on the androgen axis, while others become truly androgen receptor (AR)-independent. Our research identified the human tousled-like kinase 1 (TLK1) as a crucial early mediator of PCa cell adaptation to ADT, promoting androgen-independent growth, inhibiting apoptosis, and facilitating cell motility and metastasis. Although explicit, the growing role of TLK1 biology in PCa has remained under-represented and elusive. In this review, we aim to highlight the diverse functions of TLK1 in PCa, shed light on the molecular mechanisms underlying the transition from androgen-sensitive (AS) to an androgen-insensitive (AI) disease mediated by TLK1, and explore potential strategies to counteract this process. Targeting TLK1 and its associated signaling could prevent PCa progression to the incurable metastatic castration-resistant PCa (mCRPC) stage and provide a promising approach to treating PCa.
... This phosphorylation event promotes proper chromosomal condensation during metaphase and provides radioresistance in healthy mouse breast epithelial cells (MM3MG) and yeast [70]. TLK1B also phosphorylates the histone H3-H4 chaperones Asf1a and Asf1b, enhancing their affinity to bind to H3-H4 and enabling nucleosomal assembly during replication and damage repair [71,72]. Moreover, TLK1B phosphorylates Rad9, a component of the Rad9-Rad1-Hus1 (9-1-1) heterotrimeric complex involved in DNA repair [73]. ...
... TLKs play vital roles in various essential cellular processes, including chromatin formation, DNA replication, repair, and transcription [63,71,[124][125][126]. Studies conducted on mice and cells indicate that TLK1 and TLK2 have similar functions in developing and maintaining the genome's integrity [127]. ...
Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer (PCa) treatment, with success in developing more effective inhibitors of androgen synthesis and antiandrogens in clinical practice. However, hormone deprivation and AR ablation have caused an increase in ADT-insensitive PCas’ associated with poor prognosis. Resistance to ADT arises through various mechanisms, and most castration-resistant PCas’ still rely on the androgen axis, while others become truly androgen receptor (AR)-independent. Our research identified the human tousled-like kinase 1 (TLK1) as a crucial early mediator of PCa cell adaptation to ADT, promoting androgen-independent growth, inhibiting apoptosis, and facilitating cell motility and metastasis. Although explicit, the growing role of TLK1 biology in PCa has remained under-represented and elusive. In this review, we aim to highlight the diverse functions of TLK1 in PCa, shed light on the molecular mechanisms underlying the transition from androgen-sensitive (AS) to an androgen-insensitive (AI) disease mediated by TLK1, and explore potential strategies to counteract this process. Targeting TLK1 and its associated signaling could prevent PCa progression to the incurable metastatic castration-resistant PCa (mCRPC) stage and provide a promising approach to treating PCa.
... It was confirmed that TLK1 mainly functioned dependent on mediating the H3/H4 histone chaperone ASF1, and ASF1B is phosphorylated by TLKs during DNA replication on its C-terminal tail residues S169 and S198 [31]. TLK phosphorylation of ASF1 regulated its stability, while in vertebrates, TLK1mediated phosphorylation of several sites on the Cterminal tail of ASF1 promoted its binding affinity for the histone H3/H4 heterodimer [32]. So in LGG, does ASF1B play a regulatory role in tumour by regulating TLK1? ...
Aim
Low-grade glioma (LGG), which is the second most frequent adult brain malignancy, severely threatens patients’ health and has a high recurrence rate. Histone H3/H4 chaperone anti-silencing function 1 B (ASF1B) has a tight association with the initiation and development of tumours. The expression and regulation mechanism of ASF1B in LGG were discussed.
Methods
ASF1B expression in LGG patients as well as the association of ASF1B with overall survival and disease-free survival of LGG patients were predicted by GEPIA database. The independent prognostic value of ASF1B in LGG patients was investigated by TCGA database. RT-qPCR, together with western blot was applied for the assessment of ASF1B in LGG cell lines. After ASF1B expression was inhibited, CCK8 and colony formation assays judged cell proliferation. Flow cytometry analysis and TUNEL assay appraised cell cycle as well as apoptosis. Cell migratory and invasive capacities were measured by wound healing as well as Transwell assays. Western blot tested the expression of proliferation-, cycle-, apoptosis-, and metastasis-associated proteins. STRING and GeneMANIA database predicted the relationship between ASF1B and tousled-like kinase 1 (TLK1). ChIP assay testified the affinity of ASF1B with TLK1. Subsequently, TLK1 was overexpressed and ASF1B expression interfered, and the functional assays were executed.
Results
ASF1B was discovered to be increased in LGG tissues and cells and indicates an unfavourable prognosis for LGG patients. ASF1B was not an independent prognostic factor for LGG. ASF1B deficiency obstructed the proliferation, cell cycle as well as metastasis of LGG cells, and induced cell death, which might be realized through the interaction with TLK1.
Conclusion
The interaction between ASF1B and TLK1 promoted the malignant progression of LGG.
Key messages
TLK1 interacts with ASF1B.
Interference with ASF1B inhibits the proliferative, invasive and migratory capabilities and induces the cycle arrest, along with the apoptosis of LGG cells.
The interaction between ASF1B and TLK1 promotes the malignant progression of LGG.
... Although some other kinases (Aurora B/Ipl1 kinase) also phosphorylate H3-S10, TLK1B phosphorylation of Histone H3-S10 promotes proper chromosomal condensation during metaphase and confers radio-resistance as observed in normal mouse breast epithelial cells (MM3MG) [46]. Another direct substrate of TLK1/1B is the Histone H3-H4 chaperone Asf1a and Asf1b which can be phosphorylated on several residues at C-terminus and promotes their affinity for H3-H4 that enables nucleosomal assembly with newly synthesized DNA strand during replication and damage repair [49,53]. TLK1B is also reported to phosphorylate Rad9 of Rad9-Rad1-Hus1 (9-1-1) heterotrimeric complex that acts as a clamp for DNA polymerase during repair and to mediate checkpoint activation [54] [55]. ...
Prostate cancer metastasis
