Method for changing zeta activity of DNA polymerase

Abstract

The invention discloses a method for changing zeta activity of DNA polymerase, and belongs to the technical field of biological medicine. The method comprises the following step: changing the zeta activity of the DNA polymerase by changing phosphorylation / dephosphorylation modification of residues at positions S279, S282 and S286 of human REV3L-N70 protein, or changing the stability of the REV3L-N70, or changing the interaction between the REV3L-N70 and REV3L-C protein, or changing a Caspase-mediated REV3L-C cleavage event. The zeta activity of the DNA polymerase cannot be effectively improved through overexpression of the REV3L, but after the REV3L is knocked out, the problem, such as influence on normal cell growth, also exists, and the invention provides an effective method for inhibiting or enhancing the zeta activity of the DNA polymerase from the perspective of stabilizing or removing an REV3L activity self-inhibition mechanism.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for changing the activity of DNA polymerase ζ. Background Art

[0002] Low-fidelity translesion DNA synthesis is an important source of DNA mutations. DNA polymerase ζ is the main polymerase involved in low-fidelity translesion DNA synthesis (TLS) in eukaryotic cells. Its catalytic subunit REV3L loses its proofreading function due to the lack of 3' to 5' exonuclease activity. In yeast cells, polymerase ζ does not affect cell growth and proliferation, but the vast majority (>90%) of yeast cell mutations caused by DNA damage and more than half of spontaneous mutations depend on polymerase ζ DNA synthesis activity. In higher eukaryotic cells, polymerase ζ not only participates in DNA mutation formation, but also plays an essential biological function in cell proliferation and embryonic development.

[0003] There is a sequence-dependent protein cleavage event in the maturation of the human DNA polymerase ζ catalytic subunit REV3L protein. A highly conserved threonine aspartase TASP1 can specifically catalyze the hydrolysis of the peptide bond between aspartic acid residues 525 and glycine residues 526 of the REV3L protein, producing an amino-terminal fragment (REV3L-N70) of about 70 kDa and a carbon-terminal fragment (REV3L-C) of more than 310 kDa containing the complete polymerase domain. More than 90% of the REV3L protein in human cells exists in the form of cleavage products in a tightly interacting post-cleavage complex.

[0004] In cancer treatment, the rapid proliferation of tumor cells makes their growth and proliferation highly dependent on the replication stress relief mechanism; on the other hand, inducing cytotoxicity by replication stress is one of the main strategies for tumor chemotherapy. Translesion DNA synthesis is the main pathway for DNA damage tolerance in mammalian cells, helping to relieve replication stress. Therefore, DNA polymerase ζ activity is very likely to become an important target for improving drug sensitivity and chemotherapy resistance in tumor cells. However, in mammals, REV3L protein is indispensable for the growth, proliferation and individual development of normal cells. Downregulation of REV3L expression can significantly increase the accumulation of intracellular DNA damage and chromosomal instability, and is positively correlated with susceptibility to multiple cancers. Therefore, simply deleting REV3L expression will greatly increase the possibility of cell deterioration while improving cell sensitivity to drugs, and it also seriously interferes with normal cell growth and proliferation, all of which limit the application of DNA polymerase ζ as a target for improving drug sensitivity and resistance of tumor cells.

[0005] At the same time, DNA mutations have positive significance in specific biological processes. For example, somatic hypermutation (SHM) in the variable region of immunoglobulin genes directly determines the diversity of immunoglobulin antigen recognition. The efficient induction of somatic hypermutation in the variable region of immunoglobulin genes in vitro is the basis for establishing a full-process in vitro antibody induction and screening system. Although some work has shown that the lack of DNA polymerase ζ can significantly inhibit the occurrence of SHM in mice, simply overexpressing REV3L cannot effectively increase the activity of DNA polymerase ζ, which limits the application of DNA polymerase ζ as a target to construct a cell system that efficiently induces SHM in immunoglobulin genes. Summary of the invention

[0006] In order to solve the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for changing the activity of DNA polymerase ζ, so as to solve the problem that the prior art cannot effectively change the activity of DNA polymerase ζ.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: a method for changing the activity of DNA polymerase ζ is provided, comprising: changing the phosphorylation / dephosphorylation modification of residues S279, S282 and S286 of human REV3L-N70 protein, or changing the stability of REV3L-N70, or changing the interaction between REV3L-N70 and REV3L-C protein, or changing the Caspase-mediated REV3L-C cleavage event to change the activity of DNA polymerase ζ, that is, to enhance or weaken the activity of DNA polymerase ζ.

[0008] The beneficial effects of the present invention are:

[0009] On the basis of the above technical solution, the present invention can also be improved as follows:

[0010] Furthermore, by performing site-directed mutagenesis on residues S279, S282 and S286 of human REV3L-N70 protein, the phosphorylation / dephosphorylation modification of residues S279, S282 and S286 was enhanced or weakened, thereby changing the activity of DNA polymerase ζ.

[0011] Furthermore, the activity of DNA polymerase ζ was weakened by inhibiting the phosphorylation modification of the S279, S282 and S286 sites of the human REV3L-N70 protein by alanine substitution mutations; or the activity of DNA polymerase ζ was enhanced by simulating the continuous phosphorylation modification of the S279, S282 and S286 sites of the human REV3L-N70 protein by aspartic acid / glutamic acid substitution mutations.

[0012] Furthermore, DNA polymerase ζ activity was altered by applying replication stress to cells.

[0013] When cells face replication stress, the S279, S282 and S286 residues of REV3L-N70 protein can undergo ATR-mediated phosphorylation modification, which can increase the activity of DNA polymer ζ in cells, while inhibiting its phosphorylation modification weakens the activity of DNA polymer ζ. When cells face replication stress, the S279 cluster serine phosphorylation of REV3L-N70 protein can be induced, which can weaken the interaction between REV3L-N70 and REV3L-C, thereby changing the activity of DNA polymer ζ. Replication stress can be chemical and physical methods such as ultraviolet irradiation, hydroxyurea (HU) treatment, and cisplatin (DDP) treatment.

[0014] The sequence of the S279 serine cluster where the phosphorylation modification that affects REV3L activation is located is TSQMSQPESQD. Alanine substitution mutation at this site will inhibit its phosphorylation, while aspartic acid / glutamic acid substitution mutation at this site can simulate sustained phosphorylation.

[0015] Furthermore, under conditions of replication stress or inducing ATR kinase activation, DNA polymerase ζ activity is altered by promoting or inhibiting ATR expression or activity.

[0016] Under the conditions of applying replication pressure or inducing ATR kinase activation, siRNA or shRNA or ATR-specific small molecule inhibitors are used to inhibit the expression or activity of ATR, thereby interfering with the serine phosphorylation modification of the S279 cluster of the REV3L protein, thereby inhibiting the activation of DNA polymerase ζ under the corresponding treatment conditions, interfering with the biological function of DNA polymerase ζ in trans-damage DNA synthesis, increasing the sensitivity of cells to replication pressure caused by chemotherapeutic drugs, and reducing drug resistance caused by DNA mutations mediated by DNA polymerase ζ.

[0017] Alternatively, ATR activators or truncated fragments of ATR activating protein can be used directly to activate ATR, promote ATR-mediated REV3L phosphorylation, and thereby change DNA polymerase ζ activity.

[0018] Furthermore, upon application of replication stress, DNA polymerase ζ activity is altered by promoting or inhibiting Chk1 and PP2A expression / activity.

[0019] When cells face replication stress, residues S279, S282, and S286 on the REV3L-N70 protein can undergo ATR-mediated phosphorylation modification. The Chk1-PP2A pathway can inhibit the dephosphorylation reaction of residues S279, S282, and S286 on the REV3L-N70 protein, increase the steady-state phosphorylation modification level of the S279 cluster serine of the REV3L-N70 protein, and increase the activity of DNA polymerase ζ in cells. Specifically, siRNA, shRNA, or Chk1 small molecule inhibitors are used to inhibit the expression or activity of Chk1, inhibit PP2A activation by inhibiting Chk1-mediated PP2A phosphorylation modification, interfere with PP2A-mediated S279 cluster serine dephosphorylation reaction, increase the phosphorylation modification level of this site, and promote the activation of DNA polymerase ζ. Utilizing siRNA, shRNA or PP2A small molecule inhibitors to inhibit the expression or activity of PP2A interferes with the PP2A-mediated S279 cluster serine dephosphorylation reaction, increases the phosphorylation modification degree of this site, and promotes the activity of DNA polymerase ζ.

[0020] Furthermore, by blocking or promoting the phosphorylation / dephosphorylation modification of residues S279, S282 and S286 on the REV3L-N70 protein, the interaction between REV3L-N70 and REV3L-C proteins is promoted or blocked, thereby changing the activity of DNA polymerase ζ; or by interfering with REV3L-N70 to affect the interaction between REV3L-N70 and REV3L-C proteins, thereby changing the activity of DNA polymerase ζ.

[0021] REV3L is the catalytic subunit of DNA polymerase ζ. During the maturation of REV3L protein, there is a TASP1-mediated hydrolysis event, which produces two fragments, REV3L-N70 and REV3L-C, of ​​which REV3L-N70 is the amino-terminal cleavage fragment of REV3L protein, and REV3L-C is the carboxyl-terminal cleavage fragment of REV3L protein. REV3L protein includes 3130 amino acids in total, of which REV3L-N70 includes 525 amino acids and REV3L-C includes 2605 amino acids. REV3L-C contains a conserved DNA polymerase catalytic center, which performs the function of chain extension during DNA synthesis. The inventors found that REV3L-N70 can negatively regulate the polymerase catalytic center on REV3L-C. This mechanism can ensure that REV3L-mediated low-fidelity DNA synthesis is strictly controlled during normal cell growth. The inventors also found that the phosphorylation modification of the serine in the S279 cluster of the REV3L-N70 protein can weaken the interaction between REV3L-N70 and REV3L-C, thereby relieving the inhibition of REV3L-N70 on the polymerase catalytic active center on REV3L-C and activating DNA polymerase ζ. Therefore, the interaction between REV3L-N70 and REV3L-C can be blocked by small peptides or small molecule compounds, thereby promoting the activation of DNA polymerase ζ. The target of the small peptide or small molecule is to affect the phosphorylation / dephosphorylation modification of the S279, S282 and S286 residues of the REV3L-N70 protein, thereby affecting the interaction between REV3L-N70 and REV3L-C proteins, and ultimately affecting the activity and protein stability of DNA polymerase ζ.

[0022] In addition to the above-mentioned changes in the serine phosphorylation modification of the S279 cluster of the REV3L-N70 protein to weaken the interaction between REV3L-N70 and REV3L-C, small molecule peptides, small molecule compounds, antibodies, etc. that target the interaction interface between REV3L-N70 or REV3L-C proteins can also be used to compete or interfere with the protein interaction between REV3L-N70 and REV3L-C, thereby affecting the activity of DNA polymerase ξ.

[0023] Furthermore, the activity of DNA polymerase ζ is changed by promoting or inhibiting the expression or activity of Caspase; or the site-directed mutagenesis of the cleavage site on REV3L-C is inhibited to change the activity of DNA polymerase ζ; or the sequence element on which Caspase acts on REV3L protein is blocked to change the activity of DNA polymerase ζ; wherein the amino acid sequence of the sequence element on which Caspase acts is SQIDG.

[0024] REV3L-N70 maintains the stability of REV3L-C protein while inhibiting the catalytic activity of DNA polymerase ζ. Under replication pressure, the released REV3L-C undergoes caspase-mediated protein cleavage and protease-mediated degradation, thereby effectively limiting the action time of the activated error-prone DNA polymerase ζ and controlling the adverse effects of low-fidelity DNA synthesis mediated by DNA polymerase ζ on genome stability.

[0025] Caspase-mediated REV3L-C cleavage events can affect DNA polymerase ζ activity, so the activity of DNA polymerase ζ can be changed by changing the expression or activity of Caspase, such as by adding Caspase 3 and 7 small molecule inhibitors to inhibit Caspase-mediated REV3L-C cleavage and degradation; or by causing site-directed mutations at the cleavage site of REV3L-C to change the activity of DNA polymerase ζ, such as by causing alanine substitution mutations at the D2268 residue of the REV3L-C protein to inhibit Caspase-mediated REV3L-C cleavage to change the activity of DNA polymerase ζ. Small peptides or small molecule compounds can also be used to promote or block the sequence element of Caspase action, which is the part of REV3L-C that interacts with Caspase. By blocking this part, Caspase-mediated REV3L-C cleavage after activation can be interfered with, affecting the activity of DNA polymerase ζ.

[0026] The present invention has the following beneficial effects:

[0027] The present invention provides a method for changing the activity of DNA polymerase ζ, which can enhance or weaken the activity of DNA polymerase ζ by changing the phosphorylation modification of the serine in the S279 cluster of human REV3L protein, or the stability of REV3L-N70, or the interaction between REV3L-N70 and REV3L-C protein, or the Caspase-mediated REV3L-C cleavage event, without directly interfering with the expression level of REV3L protein.

[0028] Overexpression of REV3L cannot effectively increase the activity of DNA polymerase ζ, and knocking out REV3L will also cause problems such as affecting normal cell growth. The present invention provides an effective method for inhibiting or enhancing the activity of DNA polymerase ζ from the perspective of stabilizing or relieving the autoinhibitory mechanism of REV3L activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1ATR-mediated phosphorylation of the S279 cluster serine of the REV3L protein; Figure a shows the phosphorylation modification of the S279 cluster serine of the REV3L protein induced by replication stress caused by ultraviolet (UV), hydroxyurea (HU) and cisplatin (DDP); Figure b shows the phosphorylation modification of the S279 cluster serine of the REV3L protein after treatment with the specific ATR kinase inhibitor AZD6738.

[0030] Figure 2 The results of the dephosphorylation reaction of the S279 cluster serine of the REV3L protein mediated by the Chk1-PP2A signaling pathway; Figure a shows the phosphorylation degree of the S279 cluster serine of the REV3L protein in the cells after treatment with a specific small molecule inhibitor of Chk1; Figure b shows the phosphorylation degree of the S279 cluster serine of the REV3L protein in the cells after treatment with a specific small molecule inhibitor of PP2A; Figure c shows the phosphorylation degree of the S279 cluster serine of the REV3L protein in the cells after infection with shRNA viruses specifically targeting Chk1 and PP2A.

[0031] Figure 3 Replication stress activates REV3L-mediated DNA synthesis; Figure a shows the protein levels of REV3L-N70, REV3L-C and β-actin in the REV3L conditional knockout cell line HCT116-R-REV3L after culture in the medium with or without doxycycline; Figure b shows the DNA synthesis after continued HU treatment; Figure c shows the results of the proximity ligation experiment to detect the amount of REV3L protein recruited to active replication sites.

[0032] Figure 4 To inhibit the effect of ATR activity on the polymerase catalytic activity of REV3L; Figure a shows the effect of ATR inhibitor on the recruitment of REV3L to active replication sites after UV irradiation; Figure b shows the effect of REV3L knockout on cisplatin-induced RPA and H2AX protein phosphorylation modification levels; Figure c shows the effect of cells induced or knocked out REV3L expression after treatment with ATR kinase-specific inhibitors on cisplatin-induced RPA and H2AX protein phosphorylation modification levels.

[0033] Figure 5 The aspartate substitution mutation of the S279 cluster serine can activate the catalytic activity of REV3L polymerase; Figure a is the sequence of the REV3L-3D transcript induced to be expressed in cells in which the REV3L-3D mutant protein was induced to be expressed, and Figure b is the result of the APH-insensitive EdU incorporation experiment indicating REV3L-mediated DNA synthesis; Figure c is the result of the EdU / REV3L-proximity ligation labeling experiment indicating the recruitment of REV3L at active replication sites.

[0034] Figure 6ATR-mediated phosphorylation modification can seriously weaken the interaction between REV3L-N70 and REV3L-C; Figure a shows the effect of ultraviolet irradiation on the protein interaction between REV3L-N70 and REV3L-C; Figure b shows the effect of ultraviolet irradiation on the protein interaction between REV3L-N70 and REV3L-C after pretreatment with ATR inhibitor; Figure c shows the interaction between REV3L-N70-3D mutant protein and REV3L-C without ultraviolet irradiation.

[0035] Figure 7 The results show that REV3L-N70 negatively regulates DNA polymerase ζ activity; Figure a shows the effect of IAA treatment on the amount of REV3L protein recruited to active replication sites in cells after cisplatin treatment; Figure b shows the effect of IAA treatment on the ability of cells to alleviate cisplatin-induced replication stress and DNA damage.

[0036] Figure 8 Caspase-mediated cleavage of REV3L-C under replication stress; Figure a shows the protein levels of REV3L-C and β-actin after the cells were irradiated with UV and continued to be cultured in medium containing CHX; Figure b shows the effects of MG132 treatment and broad-spectrum caspase inhibitor z-VAD-fmk treatment on REV3L-C under replication stress; Figure c shows the effect of the D2268 residue in the Caspase 3 / 7 cleavage sequence element on the caspase-mediated cleavage of REV3L-C induced by replication stress.

[0037] Fig. 9 REV3L-N70 is involved in maintaining the stability of REV3L-C; Figure a is a schematic diagram of the REV3L gene structure in the REV3L-SP mutant cell line; Figure b shows the effect of inducing REV3L-N70 expression on the REV3L-C protein level. DETAILED DESCRIPTION

[0038] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0039] Embodiment 1:

[0040] This example is to confirm the effect of ATR expression and activity on the degree of serine phosphorylation modification of the S279 cluster of REV3L protein. The specific experimental process is as follows:

[0041] In the human colon cancer cell line HCT116, the 3xFLAG tag coding sequence was integrated upstream of the start codon of two endogenous REV3L alleles through homologous recombination technology, obtaining HCT116-3F-REV3L cells that can constitutively express human REV3L protein fused with an amino-terminal 3xFLAG tag.

[0042] In HCT116-3F-REV3L cells expressing human REV3L protein fused with 3xFLAG tag at the amino terminus, 10 J / m 2 After 4 hours of ultraviolet (UV) irradiation, 4 hours of 2mM hydroxyurea (HU), or 2 hours of 10μM cisplatin (DDP) treatment, the cells were recovered for 24 hours. The REV3L protein S279 cluster serine phosphorylation modification in whole cell lysate was evaluated by immunoblotting using a REV3L specific antibody that specifically recognizes the phosphorylated S279 cluster serine. The results showed that the replication stress caused by ultraviolet (UV), hydroxyurea (HU) and cisplatin (DDP) can specifically induce the phosphorylation modification of the REV3L protein S279 cluster serine ( Figure 1 a).

[0043] When the above treatment was performed, superimposed treatment with the specific ATR kinase inhibitor AZD6738 (0.1 μM) significantly reduced the phosphorylation of the S279 cluster serine of the REV3L protein ( Figure 1 b).

[0044] Figure 1 UC is Untreated control, DM is DMSO, and DM in other figures also represents DMSO.

[0045] Embodiment 2:

[0046] This example is to confirm the effect of Chk1 and PP2A expression and activity on the degree of serine phosphorylation modification of the S279 cluster of REV3L protein. The specific experimental process is as follows:

[0047] HCT116-3F-REV3L cells were treated with 50 J / m 2 After UV irradiation, 0.1% DMSO (DM, solvent control) or Chk1-specific small molecule inhibitors 0.5μM ChK1-124 (I1), 3μMUCN-01 (I2) and 0.1μM LY2603618 (I3) were added to continue culturing the cells for 2 hours, and then the degree of REV3L protein S279 cluster serine phosphorylation modification in the whole cell lysate was evaluated by immunoblotting. The results showed that inhibiting Chk1 activity can increase the level of REV3L protein S279 cluster serine phosphorylation in cells under replication stress ( Figure 2 a).

[0048] HCT116-3F-REV3L cells were treated with 50 J / m 2 After UV irradiation, 0.1% DMSO (DM, solvent control) or PP2A specific small molecule inhibitors 10μM LB-100 (I1) and 1μM Okadaic Acid (I2) were added and the cells were cultured for 2 hours. Then, the degree of serine phosphorylation modification of the S279 cluster of REV3L protein in the whole cell lysate was evaluated by immunoblotting. The results showed that inhibition of PP2A activity could increase the level of serine phosphorylation of the S279 cluster of REV3L protein in cells under replication stress ( Figure 2 b).

[0049] HCT116-3F-REV3L cells were infected with shRNA virus targeting Chk1 and PP2A for 3 days and then treated with 50J / m 2 After UV irradiation, the cells were cultured for 2 hours, and then the degree of serine phosphorylation modification of REV3L protein S279 cluster in the whole cell lysate was evaluated by immunoblotting. Non-targeted control shRNA (NTC) was used as a control during the experiment. The results showed that knocking down Chk1 and PP2A expression could increase the level of serine phosphorylation of REV3L protein S279 cluster in cells under replication stress ( Figure 2 c).

[0050] Embodiment 3:

[0051] This example is to confirm that DNA polymerase ζ needs replication pressure to be activated, and that simply overexpressing REV3L cannot significantly affect REV3L-mediated DNA synthesis. The specific experimental process is as follows:

[0052] Using the REV3L conditional knockout cell line and the characteristics that DNA polymerase ζ is insensitive to the B family DNA polymerase inhibitor Aphidicolin (APH), it was found that in the absence of exogenous replication pressure, doxycycline induction of REV3L expression for 2 days with (+DOX) or without (-DOX) did not significantly affect REV3L-mediated APH-insensitive EdU incorporation, indicating that overexpression of REV3L alone does not significantly affect DNA polymerase ζ activity ( Figure 3 a and 3b). Specific experimental process:

[0053] In the human colon cancer cell line HCT116, the promoters of the two endogenous REV3L alleles of the cell were replaced by a DNA fragment containing a tetracycline-inducible promoter (Ptet) and a FLAG tag coding sequence through homologous recombination technology, resulting in the cell line HCT116-R-REV3L, in which REV3L protein expression can be specifically induced or knocked out simply by adding or removing doxycycline in the culture medium.

[0054] The REV3L conditional knockout cell line HCT116-R-REV3L was cultured in the presence or absence of 20ng / mL doxycycline (DOX) for 3 days, and the protein levels of REV3L-N70, REV3L-C and β-actin in the cells were detected by immunoblotting. In the above REV3L conditional knockout cell line HCT116-R-REV3L with doxycycline induction for 3 days, 2mM HU was added (+HU) or not added (-HU) and cultured for 24 hours, and then the cells were released into the medium containing 0.5μM Aphidicolin (APH) and continued to be cultured for 20 minutes, and then 20μM EdU was added for 10 minutes. After labeling, the cells were covalently linked to the incorporated EdU molecules by Click-it reaction, and the samples were photographed by fluorescence microscopy for quantitative analysis of nuclear fluorescence signals.

[0055] Using EdU / REV3L-proximity labeling assays (PLA), which indicate REV3L recruitment to active replication sites, we demonstrated that replication stress significantly increases REV3L protein localization at active replication forks ( Figure 3 c). Specific experimental procedures: In the above REV3L conditional knockout cell line HCT116-R-REV3L in which REV3L expression was induced by doxycycline for 3 days, 2mM HU was added (+HU) or not added (-HU) and cultured for 24 hours, and then the cells were released into normal culture medium and cultured for 20 minutes, and then 20μM EdU was added for 10 minutes. After the cells were fixed, the amount of REV3L protein recruited to the active replication site was detected by Click-it reaction and proximity labeling experiment.

[0056] Embodiment 4:

[0057] This example is to confirm that inhibiting ATR activity can interfere with the polymerase catalytic activity of REV3L. The specific experimental process is as follows:

[0058] HCT116 cells were pretreated with ATR kinase specific inhibitor AZD6738 (0.1 μM) for 1 hour and then subjected to 5 J / m 2 After UV irradiation, cells were cultured for 2 hours and then labeled with 20 μM EdU for 10 minutes. After fixation, the proximity ligation assay (PLA) targeting EdU and REV3L catalytic domain (REV3L-C) was used to detect REV3L protein recruited to active replication forks. PLA experiments were performed according to the recommended procedures of Duolink PLA kit provided by Sigma. The experimental results showed that ATR inhibitor (ATRi) can significantly inhibit the recruitment of REV3L to active replication sites after UV irradiation ( Figure 4 a).

[0059] After the replication fork is stalled, a large number of single-stranded DNA regions will be generated near the replication fork, thereby inducing RPA phosphorylation; after the double-strand break occurs, the serine 139 of the H2AX protein will be rapidly phosphorylated to produce a γ-H2AX signal; therefore, the phosphorylation degree of RPA and H2AX is widely used to indicate the accumulated replication stress and DNA damage in cells. The REV3L conditional knockout cell line HCT116-R-REV3L was cultured in the medium with or without 20ng / ml doxycycline (DOX) for 3 days to induce or knock out the expression of REV3L, and then different doses of cisplatin (0, 10, 20, 40μM) were added for 2 hours ( Figure 4 b), after the cells were cultured for 24 hours, the expression of REV3L protein in the cell lysate and the phosphorylation modification of RPA and H2AX proteins were detected by immunoblotting. The results showed that the ability of human cells to alleviate cisplatin (DDP)-induced replication stress (reflected in the phosphorylation of RPA) and DNA damage (reflected in the expression level of γ-H2AX) mainly depends on the REV3L-mediated TLS pathway.

[0060] After cisplatin treatment, adding 0.1 μM ATR kinase-specific inhibitor AZD6738 to the recovery culture of cells knocked out of REV3L expression (-DOX) did not produce an additive effect, further increasing the phosphorylation levels of RPA and H2AX, indicating that ATR and REV3L are involved in alleviating cisplatin-induced replication stress and DNA damage in the same biological process ( Figure 4 c), supporting the idea that ATR is essential for the TLS polymerase catalytic activity of REV3L.

[0061] Embodiment 5:

[0062] This example is to confirm that aspartic acid substitution mutations at residues S279, S282 and S286 of REV3L protein that simulate continuous phosphorylation modification can significantly improve the catalytic activity of REV3L polymerase. The specific experimental process is as follows:

[0063] The coding sequence of residues S279, S282 and S286 of an endogenous REV3L gene was site-directedly mutated to aspartic acid codons, and the upstream endogenous promoter of the mutant REV3L-3D allele was replaced with an inducible promoter Ptet that can respond to doxycycline, thereby obtaining a cell line HCT116-REV3L-3D that can specifically induce REV3L-3D protein with aspartic acid substitution mutations at residues S279, S282 and S286 that simulates continuous phosphorylation modification, which can be specifically induced by doxycycline. Figure 5 a shows the partial sequencing results of REV3L mRNAs expressed in the corresponding mutant cell lines.

[0064] In cells inducibly expressing wild-type REV3L and REV3L-3D mutant proteins, APH-insensitive EdU incorporation assays were performed to indicate REV3L-mediated DNA synthesis ( Figure 5 b) and EdU / REV3L-proximity junction labeling experiments indicating REV3L recruitment to active replication sites ( Figure 5 c), revealing that even in the absence of exogenous replication pressure, the REV3L-3D mutant protein can still be in a polymerase catalytically active state. The corresponding experimental methods and sample processing conditions are the same as those in Example 3, except that HU to induce replication pressure is not added in this example.

[0065] Embodiment 6:

[0066] This example is to clarify that replication stress-induced REV3L protein S279 cluster serine phosphorylation can weaken the interaction between REV3L-N70 and REV3L-C. The specific experimental process is as follows:

[0067] HCT116 cells expressing REV3L protein fused with FLAG tag at the amino terminal were treated with 100 J / m 2 After the cells were cultured for another 2 hours after UV irradiation, immunoprecipitation experiments were performed using specific antibodies targeting the FLAG tag, and the abundance of each target protein enriched by the FLAG antibody was detected by immunoblotting. The experimental results showed that compared with the MK sample without UV irradiation (compared with the experimental sample, this sample was only not UV irradiated, and the rest of the process was the same), the amount of REV3L-C protein that could be co-immunoprecipitated with FLAG-REV3L in the UV-irradiated cells was significantly reduced ( Figure 6 a), indicating that UV irradiation can weaken the protein interaction between REV3L-N70 and REV3L-C.

[0068] Figure 6 In a, 3F-REV3L is a REV3L protein with a 3xFLAG tag fused to the N-terminus. It is more efficient to use the FLAG tag for N70 immunoprecipitation.

[0069] HCT116 cells expressing REV3L protein fused to the amino-terminal FLAG tag were pretreated with or without the ATR kinase specific inhibitor AZD6738 (0.1 μM) for 1 hour and then treated with 100 J / m 2 After the cells were cultured for another 2 hours after UV irradiation, immunoprecipitation and immunoblotting experiments were performed using specific antibodies targeting the FLAG tag. The results showed that 1 hour of ATR inhibitor pretreatment could significantly reduce the effect of UV irradiation on the protein interaction between REV3L-N70 and REV3L-C ( Figure 6 b).

[0070] The same immunoprecipitation and immunoblotting experiments were performed in cells expressing wild-type REV3L or REV3L-3D with aspartate substitution mutation at S279 cluster serine without UV irradiation. It was found that compared with wild-type REV3L, the interaction between REV3L-N70-3D mutant protein and REV3L-C was significantly reduced to the point of almost complete loss ( Figure 6 c).

[0071] In summary, replication stress-induced REV3L protein S279 cluster serine phosphorylation can weaken the interaction between REV3L-N70 and REV3L-C, thereby affecting DNA polymerase ζ activity.

[0072] Embodiment 7:

[0073] This example is to clarify that REV3L-N70 can negatively regulate DNA polymerase ζ activity. The specific experimental process is as follows:

[0074] Through the knock-in strategy, a cell line expressing mAID-REV3L protein with mAID degrader fused to its amino terminus was constructed in the human colon cancer cell line HCT116. After the cell line was treated with 80μM cisplatin for 3 hours, 1mM auxin indoleacetic acid (IAA) was added and cultured for another hour, inducing the rapid degradation of mAID-REV3L protein amino-terminal cleavage fragment mAID-REV3L-N70 in the cells. The fixed cells were tested by PLA experiment to detect REV3L protein on active replication forks, and it was found that IAA treatment can significantly increase the amount of REV3L protein recruited to active replication sites ( Figure 7 a), indicating that IAA-induced degradation of mAID-REV3L-N70 can enhance REV3L-mediated DNA synthesis.

[0075] Cell lines expressing wild-type REV3L or mAID-REV3L protein were treated with 80 μM cisplatin for 3 hours and then recovered in the presence or absence of 1 mM IAA for 24 hours. The accumulated RPA and H2AX phosphorylation levels in the cells were then detected by immunoblotting. The experimental results showed that compared with cells expressing wild-type REV3L, IAA treatment specifically reduced the phosphorylation of RPA and H2AX proteins induced by cisplatin in cells expressing mAID-REV3L protein, indicating that IAA-induced degradation of mAID-REVL-N70 can specifically enhance the ability of cells to alleviate cisplatin-induced replication stress and DNA damage ( Figure 7 b), supporting the idea that REV3L-N70 negatively regulates the catalytic activity of REV3L polymerase.

[0076] Embodiment 8:

[0077] This example is to clarify that Caspase-mediated cleavage of REV3L-C occurs under replication stress, and REV3L-N70 participates in maintaining the stability of REV3L-C. The specific experimental process is as follows:

[0078] HCT116-R-REV3L cells were treated with 50 J / m 2 After UV irradiation, 100 μM cycloheximide (CHX, a protein translation inhibitor) was added and treated for different time periods, and the expression of the target protein in the cell lysate was detected. The results showed that the half-life of REV3L-C carrying the conserved polymerase catalytic domain decreased significantly after UV irradiation ( Figure 8 a, Figure 8 MOCK in a is a negative control).

[0079] Cells were exposed to 50 J / m 2 After treatment with UV, 80 μM cisplatin, or 2 mM hydroxyurea (HU), the cells were cultured for 6 hours in a medium containing 0.1% DMSO (solvent control) or 10 μM proteasome inhibitor MG132, and immunoblotting was performed to detect the proteins shown. It was found that MG132 treatment could stabilize the REV3L-C cleavage band of about 90 kD (called C90 protein) ( Figure 8 b, lanes 3, 5, and 7). However, the addition of the broad-spectrum caspase inhibitor z-VAD-fmk (25 μM) while treating with MG132 completely inhibited the appearance of drug-induced C90 ( Figure 8 b, lanes 4, 6, and 8 compared with lanes 3, 5, and 7, respectively), indicating that Caspase-mediated cleavage of REV3L-C occurs under replication stress, and the C90 produced by cleavage can be further degraded by the proteasome.

[0080] The sequence elements in the REV3L-C coding sequence that affect C90 formation were identified through site-directed mutagenesis and transient expression in HCT116 cells. It was found that after the alanine substitution mutation of D2268 in the potential Caspase 3 / 7 cleavage sequence element SQIDG (2265a.a.-2269a.a.), the cells were exposed to 50J / m 2 After UV irradiation, the C90 fragment derived from the REV3L-D2268A mutant protein did not appear ( Figure 8 c), indicating that residue D2268 in the SQIDG sequence element is essential for replication stress-induced caspase-mediated cleavage of REV3L-C.

[0081] Embodiment 9:

[0082] This example is to clarify that REV3L-N70 participates in maintaining the stability of REV3L-C. The specific experimental process is as follows:

[0083] The inducible promoter P was integrated upstream of the endogenous REV3L gene start coding sequence and G526 coding sequence by knock-in strategy. tet and the constitutive promoter P CMV In HCT116 cells, a cell line HCT117-R-REV3L-SP was constructed that can induce REV3L-N70 expression while continuously expressing REV3L-C ( Fig. 9 a). After the cells were cultured for 2 days in the presence or absence of 1 μg / ml doxycycline, the amino-terminal (N70) and carboxyl-terminal cleavage fragments (REV3L-C) of REV3L were detected by immunoblotting. It was found that induction of REV3L-N70 could significantly increase the steady-state REV3L-C protein level in the cells ( Fig. 9 b), indicating that REV3L-N70 is involved in maintaining REV3L-C stability.

[0084] Fig. 9 MCM3 in the assay was used as an internal reference to normalize the differences in sample loading between samples. Fig. 9 b The left side is the RT-PCR graph, and the right side is the Western graph. By combining the left and right sides, it can be seen that the transcription amount of REV3L-C gene is almost the same with or without induction of N70 expression, but the REV3L-C protein can only exist stably when N70 is expressed at the same time.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for changing the activity of DNA polymerase ζ, characterized in that: include: The DNA polymerase ζ activity is altered by changing the phosphorylation / dephosphorylation modification of residues S279, S282 and S286 of the human REV3L-N70 protein, or by changing the stability of REV3L-N70, or by changing the interaction between REV3L-N70 and REV3L-C protein, or by changing the Caspase-mediated REV3L-C cleavage event.

2. The method for changing the activity of DNA polymerase ζ according to claim 1, characterized in that: By performing site-directed mutagenesis on residues S279, S282 and S286 of human REV3L-N70 protein, the phosphorylation / dephosphorylation modification of residues S279, S282 and S286 was enhanced or weakened, thereby changing the DNA polymerase ζ activity.

3. The method for changing the activity of DNA polymerase ζ according to claim 2, characterized in that: The activity of DNA polymerase ζ was weakened by inhibiting phosphorylation modification at the S279, S282 and S286 sites of the human REV3L-N70 protein through alanine substitution mutations; or the activity of DNA polymerase ζ was enhanced by simulating continuous phosphorylation modification at the S279, S282 and S286 sites of the human REV3L-N70 protein through aspartic acid / glutamic acid substitution mutations.

4. The method for changing the activity of DNA polymerase ζ according to claim 1, characterized in that: This also includes altering DNA polymerase zeta activity by imposing replication stress on cells.

5. The method for changing the activity of DNA polymerase ζ according to claim 4, characterized in that: Under conditions that impose replication stress or induce ATR kinase activation, DNA polymerase ζ activity is altered by promoting or inhibiting ATR expression or activity.

6. The method for changing the activity of DNA polymerase ζ according to claim 5, characterized in that: The activity of DNA polymerase ζ is changed by inhibiting the expression or activity of ATR using siRNA, shRNA or ATR inhibitor; or by activating ATR using ATR activator or ATR activation protein truncated fragment.

7. The method for changing the activity of DNA polymerase ζ according to claim 4, characterized in that: Under applied replication stress, DNA polymerase ζ activity is altered by promoting or inhibiting Chk1 and PP2A expression / activity.

8. The method for changing the activity of DNA polymerase ζ according to claim 7, characterized in that: The activity of DNA polymerase ζ is changed by inhibiting the expression or activity of Chk1 using siRNA, shRNA or Chk1 inhibitor; or the expression or activity of PP2A is changed by inhibiting the expression or activity of PP2A using siRNA, shRNA or PP2A inhibitor.

9. The method for changing the activity of DNA polymerase ζ according to claim 1, characterized in that: The activity of DNA polymerase ζ is changed by promoting or blocking the interaction between REV3L-N70 and REV3L-C proteins by blocking or promoting the phosphorylation / dephosphorylation modification of residues S279, S282 and S286 on the REV3L-N70 protein; or by affecting the interaction between REV3L-N70 and REV3L-C proteins by interfering with competition with REV3L-N70, thereby changing the activity of DNA polymerase ζ.

10. The method for changing the activity of DNA polymerase ζ according to claim 1, characterized in that: The activity of DNA polymerase ζ is changed by promoting or inhibiting the expression or activity of Caspase; or the site-directed mutation of the cleavage site on REV3L-C is inhibited to change the activity of DNA polymerase ζ; or the sequence element on which Caspase acts on REV3L protein is blocked to change the activity of DNA polymerase ζ; wherein the amino acid sequence of the sequence element on which Caspase acts is SQIDG.

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