IgG is an aging factor that drives adipose tissue fibrosis and metabolic decline.

Aging is underpinned by pronounced metabolic decline; however, the drivers remain obscure. Here, we report that IgG accumulates during aging, particularly in white adipose tissue (WAT), to impair adipose tissue function and metabolic health. Caloric restriction (CR) decreases IgG accumulation in WAT, whereas replenishing IgG counteracts CR's metabolic benefits. IgG activates macrophages via Ras signaling and consequently induces fibrosis in WAT through the TGF-ß/SMAD pathway. Consistently, B cell null mice are protected from aging-associated WAT fibrosis, inflammation, and insulin resistance, unless exposed to IgG. Conditional ablation of the IgG recycling receptor, neonatal Fc receptor (FcRn), in macrophages prevents IgG accumulation in aging, resulting in prolonged healthspan and lifespan. Further, targeting FcRn by antisense oligonucleotide restores WAT integrity and metabolic health in aged mice. These findings pinpoint IgG as a hidden culprit in aging and enlighten a novel strategy to rejuvenate metabolic health.

Association between serum uric acid and homocysteine levels among adults in the United States: a cross-sectional study.

BACKGROUND: Many studies have shown that both elevated serum uric acid (SUA) levels and hyperhomocysteinemia are risk factors for atherosclerosis. However, the relationship between the two has not been thoroughly investigated. OBJECTIVE: This study aimed to explore the possible link between SUA levels and homocysteine (Hcy) levels. METHODS: In this cross-sectional study, 17,692 adults aged > 19 years in National Health and Nutrition Examination Survey from 1999 to 2006 were analyzed. Multivariable linear regression analysis was performed to assess the association between SUA and Hcy levels. In addition, smooth curve fitting (penalized spline method) and threshold effect analysis were performed. RESULTS: Multivariable linear analysis showed that Hcy levels increased by 0.48 µmol/L (ß = 0.48, 95%CI: 0.43-0.53) for every 1 mg/dL increase in SUA levels. We found a nonlinear relationship between SUA and Hcy levels. The results of threshold effect analysis showed that the inflection point for SUA levels was 7.1 mg/dL (ß = 0.29, 95% CI: 0.23-0.36 and ß = 1.05, 95% CI: 0.67-1.43 on the left and right sides of the inflection point, respectively). The p-values was less than 0.001 when using the log likelihood ratio test. This nonlinear relationship was also found in both sexes. The inflection point for SUA levels was 5.4 mg/dL in males and 7.3 mg/dL in females, respectively. CONCLUSIONS: This cross-sectional study showed that the SUA levels were positively correlated with Hcy levels. And we found a nonlinear relationship between SUA and Hcy levels.

A noncanonical IRAK4-IRAK1 pathway counters DNA damage-induced apoptosis independently of TLR/IL-1R signaling.

Interleukin-1 receptor (IL-1R)-associated kinases (IRAKs) are core effectors of Toll-like receptors (TLRs) and IL-1R in innate immunity. Here, we found that IRAK4 and IRAK1 together inhibited DNA damage-induced cell death independently of TLR or IL-1R signaling. In human cancer cells, IRAK4 was activated downstream of ATR kinase in response to double-strand breaks (DSBs) induced by ionizing radiation (IR). Activated IRAK4 then formed a complex with and activated IRAK1. The formation of this complex required the E3 ubiquitin ligase Pellino1, acting structurally but not catalytically, and the activation of IRAK1 occurred independently of extracellular signaling, intracellular TLRs, and the TLR/IL-1R signaling adaptor MyD88. Activated IRAK1 translocated to the nucleus in a Pellino2-dependent manner. In the nucleus, IRAK1 bound to the PIDD1 subunit of the proapoptotic PIDDosome and interfered with platform assembly, thus supporting cell survival. This noncanonical IRAK signaling pathway was also activated in response to other DSB-inducing agents. The loss of IRAK4, of IRAK4 kinase activity, of either Pellino protein, or of the nuclear localization sequence in IRAK1 sensitized p53-mutant zebrafish to radiation. Thus, the findings may lead to strategies for overcoming tumor resistance to conventional cancer treatments.

Inactive PARP1 causes embryonic lethality and genome instability in a dominant-negative manner.

PARP1 (poly-ADP ribose polymerase 1) is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A, Parp1+/A) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1 Parp1+/A mice are chimeras with mixed Parp1+/AN (neoR retention) cells that act similarly to Parp1+/-. Pure F2 Parp1+/A embryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared to Parp1-/- cells, genotype and expression-validated pure Parp1+/A cells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatid exchange and mitotic bridges. Despite proficiency for homologous recombination and nonhomologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development, Parp1+/A cells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity of Parp1+/A cells to base damages and Topo inhibitors exceed Parp1-/- controls. The findings show that the enzymatically inactive PARP1 dominant negatively blocks DNA repair in selective pathways beyond wild-type PARP1 and establishes a crucial physiological difference between PARP1 inactivation vs. deletion. As a result, the expression of enzymatically inactive PARP1 from one allele is sufficient to abrogate murine embryonic development, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy.

A Non-Canonical IRAK Signaling Pathway Triggered by DNA Damage.

Interleukin-1 receptor (IL-1R)-associated kinases (IRAKs) are core effectors of Toll-like receptor (TLR) and IL-1R signaling, with no reported roles outside of innate immunity. We find that vertebrate cells exposed to ionizing radiation (IR) sequentially activate IRAK4 and IRAK1 through a phosphorylation cascade mirroring that induced by TLR/IL-1R, resulting in a potent anti-apoptotic response. However, IR-induced IRAK1 activation does not require the receptors or the IRAK4/1 adaptor protein MyD88, and instead of remaining in the cytoplasm, the activated kinase is immediately transported to the nucleus via a conserved nuclear localization signal. We identify: double-strand DNA breaks (DSBs) as the biologic trigger for this pathway; the E3 ubiquitin ligase Pellino1 as the scaffold enabling IRAK4/1 activation in place of TLR/IL-1R-MyD88; and the pro-apoptotic PIDDosome (PIDD1-RAIDD-caspase-2) as a critical downstream target in the nucleus. The data delineate a non-canonical IRAK signaling pathway derived from, or ancestral to, TLR signaling. This DSB detection pathway, which is also activated by genotoxic chemotherapies, provides multiple actionable targets for overcoming tumor resistance to mainstay cancer treatments.

DNA damage-induced phosphorylation of CtIP at a conserved ATM/ATR site T855 promotes lymphomagenesis in mice.

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)-mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ-mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4-/-Tp53-/- mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs-/-Tp53-/- mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ-mediated chromosomal translocation.

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair.

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase ß and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase ß and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.

FATC Domain Deletion Compromises ATM Protein Stability, Blocks Lymphocyte Development, and Promotes Lymphomagenesis.

Ataxia-telangiectasia mutated (ATM) kinase is a master regulator of the DNA damage response, and loss of ATM leads to primary immunodeficiency and greatly increased risk for lymphoid malignancies. The FATC domain is conserved in phosphatidylinositol-3-kinase-related protein kinases (PIKKs). Truncation mutation in the FATC domain (R3047X) selectively compromised reactive oxygen species-induced ATM activation in cell-free assays. In this article, we show that in mouse models, knock-in ATM-R3057X mutation (Atm⁠ RX ⁠, corresponding to R3047X in human ATM) severely compromises ATM protein stability and causes T cell developmental defects, B cell Ig class-switch recombination defects, and infertility resembling ATM-null. The residual ATM-R3057X protein retains minimal yet functional measurable DNA damage-induced checkpoint activation and significantly delays lymphomagenesis in Atm⁠ RX/RX ⁠ mice compared with Atm⁠ -/- ⁠. Together, these results support a physiological role of the FATC domain in ATM protein stability and show that the presence of minimal residual ATM-R3057X protein can prevent growth retardation and delay tumorigenesis without restoring lymphocyte development and fertility.

The plié by DNA-PK: dancing on DNA.

In this issue of Molecular Cell, Chen et al. (2020) report the structural transition during DNA-dependent activation of DNA-PK, shedding light on the mechanism by which kinase inhibitors and auto-phosphorylation-deficient DNA-PKcs compromise non-homologous end-joining (Chen et al., 2020).

CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining.

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo.

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination.

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology-all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis.

The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.

Phosphorylation at S2053 in Murine (S2056 in Human) DNA-PKcs Is Dispensable for Lymphocyte Development and Class Switch Recombination.

The classical nonhomologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large DNA-PK catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs-/- ) abrogates end processing (e.g., hairpin opening), but not end-ligation, whereas expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD ) abrogates end-ligation, suggesting a kinase-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs-/- and DNA-PKcsKD/KD mice because of the requirement for both hairpin opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056 cluster is the best-characterized autophosphorylation site in human DNA-PKcs. In this study, we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053. We also generated knockin mouse models with alanine- (DNA-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD mice retained normal kinase activity and underwent efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053 cluster of murine DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.

CtIP is essential for early B cell proliferation and development in mice.

B cell development requires efficient proliferation and successful assembly and modifications of the immunoglobulin gene products. CtIP is an essential gene implicated in end resection and DNA repair. Here, we show that CtIP is essential for early B cell development but dispensable in naive B cells. CtIP loss is well tolerated in G1-arrested B cells and during V(D)J recombination, but in proliferating B cells, CtIP loss leads to a progressive cell death characterized by ATM hyperactivation, G2/M arrest, genomic instability, and 53BP1 nuclear body formation, indicating that the essential role of CtIP during proliferation underscores its stage-specific requirement in B cells. B cell proliferation requires phosphorylation of CtIP at T847 presumably by CDK, but not its interaction with CtBP or Rb or its nuclease activity. CtIP phosphorylation by ATM/ATR at T859 (T855 in mice) promotes end resection in G1-arrested cells but is dispensable for B cell development and class switch recombination, suggesting distinct roles for T859 and T847 phosphorylation in B cell development.

Kinase-dead ATR differs from ATR loss by limiting the dynamic exchange of ATR and RPA.

ATR kinase is activated by RPA-coated single-stranded DNA (ssDNA) to orchestrate DNA damage responses. Here we show that ATR inhibition differs from ATR loss. Mouse model expressing kinase-dead ATR (Atr+/KD), but not loss of ATR (Atr+/-), displays ssDNA-dependent defects at the non-homologous region of X-Y chromosomes during male meiosis leading to sterility, and at telomeres, rDNA, and fragile sites during mitosis leading to lymphocytopenia. Mechanistically, we find that ATR kinase activity is necessary for the rapid exchange of ATR at DNA-damage-sites, which in turn promotes CHK1-phosphorylation. ATR-KD, but not loss of ATR, traps a subset of ATR and RPA on chromatin, where RPA is hyper-phosphorylated by ATM/DNA-PKcs and prevents downstream repair. Consequently, Atr+/KD cells have shorter inter-origin distances and are vulnerable to induced fork collapses, genome instability and mitotic catastrophe. These results reveal mechanistic differences between ATR inhibition and ATR loss, with implications for ATR signaling and cancer therapy.

Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD ) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD , but not DNA-PKcs-/- , B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

PAXX promotes KU accumulation at DNA breaks and is essential for end-joining in XLF-deficient mice.

Non-homologous end-joining (NHEJ) is the most prominent DNA double strand break (DSB) repair pathway in mammalian cells. PAXX is the newest NHEJ factor, which shares structural similarity with known NHEJ factors-XRCC4 and XLF. Here we report that PAXX is dispensable for physiological NHEJ in otherwise wild-type mice. Yet Paxx-/- mice require XLF and Xlf-/- mice require PAXX for end-ligation. As such, Xlf-/-Paxx-/- mice display severe genomic instability and neuronal apoptosis, which eventually lead to embryonic lethality. Despite their structural similarities, only Xlf-/- cells, but not Paxx-/- cells require ATM/DNA-PK kinase activity for end-ligation. Mechanistically, PAXX promotes the accumulation of KU at DSBs, while XLF enhances LIG4 recruitment without affecting KU dynamics at DNA breaks in vivo. Together these findings identify the molecular functions of PAXX in KU accumulation at DNA ends and reveal distinct, yet critically complementary functions of PAXX and XLF during NHEJ.

The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.

CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks.

CtIP (CtBP-interacting protein) associates with BRCA1 and the Mre11-Rad50-Nbs1 (MRN) complex and plays an essential role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair. It has been described that CtIP forms dimers in mammalian cells, but the biological significance is not clear. In this study, we identified a conserved motif in the N terminus of CtIP, which is required for dimer formation. We further showed that CtIP mutants impaired in forming dimers are strongly defective in HR, end resection, and activation of the ataxia telangiectasia and Rad3-related pathway, without notable change of CtIP interactions with BRCA1 or Nbs1. In addition to HR, CtIP dimerization is also required for microhomology-mediated end joining. Live cell imaging of enhanced GFP-tagged CtIP demonstrates that the CtIP dimerization mutant fails to be localized to DSBs, whereas placing a heterologous dimerization motif to the dimerization mutant restores CtIP recruitment to DSBs. These studies suggest that CtIP dimer formation is essential for its recruitment to DSBs on chromatin upon DNA damage. Furthermore, DNA damage-induced phosphorylation of CtIP is significantly reduced in the CtIP dimerization mutants. Therefore, in addition to the C-terminal conserved domains critical for CtIP function, the dimerization motif on the N terminus of CtIP is also conserved and essential for its function in DNA damage responses. The severe repair defects of CtIP dimerization mutants are likely due to the failure in localization to chromosomal DSBs upon DNA damage.