Chemical Genetics Reveals a Specific Requirement for Cdk2 Activity in the DNA Damage Response and Identifies Nbs1 as a Cdk2 Substrate in Human Cells

<div><p>The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2^as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1—a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling—dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR–sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.</p> </div

Ser432 is phosphorylated in S phase after Nbs1 recruitment to chromatin.

<p>(A) Time course of release from contact inhibition (G0) of RPE-hTERT cells, monitored by flow cytometry for DNA content and immunoblotting for accumulation of cyclin A, activated (Thr160-phosphorylated) Cdk2 and Ser432-phosphorylated Nbs1. (B) RPE-hTERT cells were harvested and fractionated at indicated times after release from G0. Recruitment of Nbs1 to chromatin precedes phosphorylation on Ser432. (C) RPE-hTERT cells treated with DMSO, 4 µg/ml aphidicolin or 20 µM roscovitine at indicated times after release from G0 were monitored for DNA content (<i>left</i>) and accumulation of cyclin A, activated Cdk2 and phosphorylated Nbs1 (<i>right</i>). Asterisk denotes anti-phospho-Ser432 cross-reactive ∼110 kDa band that is absent in G0 and roscovitine-sensitive, but unlikely to be an <i>Nbs1</i> gene product, because it is present in NBS-T cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002935#pgen.1002935.s002" target="_blank">Figure S2B</a>) and absent in Nbs1 immunoprecipitates (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002935#pgen-1002935-g003" target="_blank">Figure 3D, 3E</a>). (D) <i>Cdk2^as/as</i> or wild-type RPE-hTERT cells were released from G0 for indicated times in the presence of 0.5 µM 6-BAP or 10 µM 3-MB-PP1, as indicated, and tested for total (top) and Ser432-phosphorylated Nbs1 (bottom).</p

Nbs1-Ser432 is required for normal X-ray resistance but not viability.

<p>(A) Cells were treated with <i>Nbs1</i> or control siRNA and transiently transfected with empty vector or vectors encoding wild-type, S432A or S432D Nbs1 variants, as indicated, and tested for colony-forming ability. Error bars indicate +/− SD of duplicate measurements. (B) Nbs1 was immunoprecipitated from extracts of NBS-T cells, untransfected or stably expressing wild-type, S432A or S432D <i>Nbs1</i>, and probed for Nbs1, Rad50 and Mre11. (C) IR sensitivity of NBS-T cells expressing indicated Nbs1 isoforms. Cells were irradiated at indicated doses and tested for colony formation 14 d after irradiation. (D) IR sensitivity of cells from (C), transiently transfected with siRNA targeting <i>Nbs1</i>. (The parental NBS-T cells that express no full-length Nbs1 do not survive this treatment.) Values represent the means of duplicates +/− SD.</p

Cdk2 activity is required for normal DNA damage resistance.

<p>(A) Transient treatment with 10 µM 3-MB-PP1 for 48 hr does not affect efficiency of colony formation by wild-type or <i>Cdk2^as/as</i> RPE-hTERT cells. (B) Treatment with 10 µM 3-MB-PP1 before and after irradiation (24 hr each) sensitizes <i>Cdk2^as/as</i> RPE-hTERT cells to killing by IR. (C) Pre-treatment with 3-MB-PP1 for 24 hr does not sensitize <i>Cdk2^as/as</i> cells to IR. (D) Treatment with 3-MB-PP1 for 24 hr after IR sensitizes <i>Cdk2^as/as</i> cells to killing. Error bars are +/− standard deviation (SD) of duplicates.</p

Nbs1 is phosphorylated by Cdk2 on Ser432 in human whole-cell extracts.

<p>(A) Labeling by Cdk2^as/or Cdk2^WT/cyclin A, as indicated, with [γ-³²P]<i>N6</i>-(benzyl)-ATP in RPE-hTERT or HCT116 whole cell extracts. (B) Anti-Mre11 immunoprecipitates from labeling reactions analyzed by autoradiography (top) and anti-Nbs1 and -Mre11 immunoblot (bottom). Arrow indicates band at position of Nbs1, which did not appear in mock immunoprecipitates lacking antibody (“control”). (C) Anti-Nbs1 immunoprecipitates from labeling reactions analyzed by autoradiography (top) and anti-Nbs1 immunoblot (bottom). (D) Reticulocyte lysates programmed with indicated cDNAs were labeled by Cdk2^as. Labeling in extract (left) and anti-Nbs1 immunoprecipitates (right) was detected by autoradiography, and expression of Nbs1 isoforms confirmed by immunoblot (bottom). (E) NBS-T cells were transiently transfected with empty vector or ones encoding Myc-tagged Nbs1 variants, as indicated. Labeling by Cdk2^as was detected by autoradiography of extract (left) and anti-Mre11 immunoprecipitates (right), and equal expression and recovery of Nbs1 isoforms were confirmed by immunoblot (bottom). Nbs1+ denotes control cells expressing full-length Nbs1 endogenously.</p

Specialized roles of Cdk2 in DNA damage response: a function of activation pathway insulation?

<p>We propose that a specific requirement for Cdk2 activity to protect cells from IR reflects its distinct mode of activation. Cdk2 is phosphorylated as a monomer by CAK, and then binds cyclin A to become active. Cdk1, in contrast, can only be phosphorylated by mammalian CAK in the presence of a cyclin (A or B), and only forms stable complexes with cyclins upon T-loop phosphorylation. This effectively couples Cdk1-activating phosphorylation to inhibitory phosphorylation by kinases such as Wee1 that also require a CDK/cyclin complex substrate <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002935#pgen.1002935-Larochelle1" target="_blank">[27]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002935#pgen.1002935-Coulonval1" target="_blank">[66]</a>, and makes Cdk1 intrinsically more sensitive to restraint by DNA damage checkpoints. By evading that restraint, Cdk2 might take the lead role in phosphorylating Nbs1-Ser432 (and possibly other targets such as CtIP) early in S phase.</p

A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, <i>RTEL1</i>, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome

<div><p>Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in <i>RTEL1</i>, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of <i>RTEL1</i>. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, <i>RTEL1</i> mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in <i>RTEL1</i> as the underlying basis of the clinical and cellular phenotypes. This study further implicates <i>RTEL1</i> in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in <i>RTEL1</i>.</p></div

Inhibiting DNA replication blocks T-circle formation in MSK-41 <i>RTEL1^R1264H</i> cells.

<p>(A) Phi29-dependent T-circles in BJ hTERT and MSK-41. (B) Phi29-dependent T-circles in RTEL1 floxed/- MEFs ± Cre, BJ hTERT and MSK-41. (C) Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (D) Dot blot of the Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (E) Quantification of the fold increase in intensity of Phi29-dependent T-circles in the different cell lines subjected to the indicated treatments. Intensity mean and standard deviation were calculated over two independent experiments; statistical analysis (one-way ANOVA) was calculated with Prism (GraphPad).</p

<i>RTEL1^R1264H</i> affects a putative conserved C4C4 domain.

<p>As displayed on the schematic (representing ENSP00000353332), the RTEL1 mutation is at the C-terminus of the protein, distal to the helicase domain. The affected amino acid is in a putative C4C4 domain. All eight key cysteines and R1264 are conserved in human, orangutan, cattle, and mouse sequences. Higher percent identity at a given amino acid position is indicated by a deeper purple color.</p

NCI-318 and MSK-41 pedigrees with <i>RTEL1</i> mutation and shared risk haplotype.

<p>NCI-318 (A) and MSK-41 (B) pedigrees are shown. Red symbols indicate affected individuals. The pink rectangles indicate the shared haplotype between the pedigrees. Each other colored rectangle indicates a unique haplotype.</p