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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+/A. 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. [ABSTRACT FROM AUTHOR]

The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters--PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf-/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQR Xlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQR Xlf-/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation. [ABSTRACT FROM AUTHOR]

Poly (ADP-ribose) polymerase-1 (PARP1) and 2 (PARP2) are two DNA damage-induced poly (ADP-ribose) (PAR) polymerases in cells and are the targets of PARP inhibitors used for cancer therapy. Strand breaks recruit and activate PARP1 and 2, which rapidly generate PAR from NAD+. PAR promotes the recruitment of other repair factors, relaxes chromatin, and has a role in DNA repair, transcription regulation, and RNA biology. Four PARP1/2 dual inhibitors are currently used to treat BRCA-deficient breast, ovarian, prostate, and pancreatic cancers. In addition to blocking the enzymatic activity of PARP1 and 2, clinical PARP inhibitors extend the appearance of PARP1 and PARP2 on chromatin after damage, termed trapping. Loss of PARP1 confers resistance to PARP inhibitors, suggesting an essential role of trapping in cancer therapy. Yet, whether the persistent PARP1 and 2 foci at the DNA damage sites are caused by the retention of the same molecules or by the continual exchange of different molecules remains unknown. Here, we discuss recent results from quantitative live-cell imaging studies focusing on PARP1 and PARP2’s distinct DNA substrate specificities and modes of recruitment and trapping with implications for cancer therapy and on-target toxicities of PARP inhibitors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-Se 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. [ABSTRACT FROM AUTHOR]

Explanations for the occurrence of hysteresis (asynchronicity) between diel soil respiration (Rs) and soil temperature (Ts) have evoked both biological and physical mechanisms. The specifics of these explanations, however, tend to vary with the particular ecosystem or biome being investigated. So far, the relative degree of control of biological and physical processes on hysteresis is not clear for drylands. This study examined the seasonal variation in diel hysteresis and its biological control in a desert-shrub ecosystem in northwest (NW) China. The study was based on continuous measurements of Rs, air temperature (Ta), temperature at the soil surface and below (Tsurf and Ts), volumetric soil water content (SWC), and photosynthesis in a dominant desert shrub (i.e., Artemisia ordosica) over an entire year in 2013. Trends in diel Rs were observed to vary with SWC over the growing season (April to October). Diel variations in Rs were more closely associated with variations in Tsurf than with photosynthesis as SWC increased, leading to Rs being in phase with Tsurf, particularly when SWC> 0.08m³ m-3 (ratio of SWC to soil porosityD0.26). However, as SWC decreased below 0.08m³ m-3, diel variations in Rs were more closely related to variations in photosynthesis, leading to pronounced hysteresis between Rs and Tsurf. Incorporating photosynthesis into a Q10-function eliminated 84.2% of the observed hysteresis, increasing the overall descriptive capability of the function. Our findings highlight a high degree of control by photosynthesis and SWC in regulating seasonal variation in diel hysteresis between Rs and temperature. [ABSTRACT FROM AUTHOR]

Bcl11b is a transcription factor important for T cell development and also a tumor-suppressor gene that is hemizygously inactivated in ∼10 % human T cell acute lymphoblastic leukemia (T-ALL) and several murine T-ALL models, including ATM-/- thymic lymphomas. Here we report that heterozygous loss of Bcl11b (Bcl11b+/-) unexpectedly reduced lethal thymic lymphoma in ATM-/- mice by suppressing lymphoma progression, but not initiation. The suppression was associated with a T cell-mediated immune response in ATM-/-Bcl11b+/- mice, revealing a haploid insufficient function of Bcl11b in immune modulation against lymphoma and offering an explanation for the complex relationship between Bcl11b status with T-ALL prognosis. [ABSTRACT FROM AUTHOR]

Intramural hematoma (IMH) is a newly defined disease entity and the optimal management is still controversial as the disease shows varied clinical course. We present a case of type B IMH, initially presenting with paraplegia progressing to segmental aortic dissection (SAD) which the formed dissection displayed as a segmental distribution pattern. To our knowledge, it may become a new progression pattern of IMH progression. The SAD was successfully treated with both thoracic and abdominal endovascular aortic repair (TEVAR plus EVAR). In 1-year follow-up, the patient recovered almost completely with moderately neurological deficit and the blood pressure is in control. [ABSTRACT FROM AUTHOR]

Nonhomologous end joining (NHEJ), a major pathway of DNA double-strand break (DSB) repair, is required during lymphocyte development to resolve the programmed DSB5 generated during Variable, Diverse, and Joining [V(D)J] recombination. XRCC4-like factor (XLF) (also called Cernunnos or NHEJ1) is a unique component of the NHEJ pathway. Although germ-line mutations of other NHEJ factors abrogate lymphocyte development and lead to severe combined immunodeficiency (SCID), XLF mutations cause a progressive lymphocytopenia that is generally less severe than SCID. Accordingly, XLF-deficient murine lymphocytes show no measurable defects in V(D)J recombination. We reported earlier that ATM kinase and its substrate histone H2AX are both essential for V(D)J recombination in XLF-deficient lymphocytes, despite moderate role in V(D)J recombination in WT cells. p53-binding protein 1 (53BP1) is another substrate of ATM. 53BP1 deficiency led to small reduction of peripheral lymphocyte number by compromising both synapse and end-joining at modest level during V(D)J recombination. Here, we report that 53BP1/XLF double deficiency blocks lymphocyte development at early progenitor stages, owing to severe defects in end joining during chromosomal V(D)J recombination. The unrepaired DNA ends are rapidly degraded in 53BP1-/-XLF-/- cells, as reported for H2AX-/-XLF-/- cells, revealing an end protection role for 53BP1 reminiscent of H2AX. In contrast to the early embryonic lethality of H2AX-/-XLF-/- mice, 53BP1-/-XLF-/- mice are born alive and develop thymic lymphomas with translocations involving the T-cell receptor loci. Together, our findings identify a unique function for 53BP1 in end-joining and tumor suppression. [ABSTRACT FROM AUTHOR]

Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI-induced DSBs in XRCC4-deficient pro-B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered. [ABSTRACT FROM AUTHOR]

Antigen receptor variable region exons are assembled during lymphocyte development from variable (V), diversity (D), and joining (J) gene segments. Each germ-line gene segment is flanked by recombination signal sequences (RSs). Recombination-activating gene endonuclease initiates V(D)J recombination by cleaving a pair of gene segments at their junction with flanking RSs to generate covalently sealed (hairpinned) coding ends (CEs) and blunt 5'-phosphorylated RS ends (SEs). Subsequently, nonhomologous end joining (NHEJ) opens, processes, and fuses CEs to form coding joins (CJs) and precisely joins SEs to form signal joins (SJs). DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activates Artemis endonuclease to open and process hairpinned CEs before their fusion into CJs by other NHEJ factors. Although DNA-PKcs is absolutely required for CJs, SJs are formed to variable degrees and with variable fidelity in different DNA-PKcs-deficient cell types. Thus, other factors may compensate for DNA-PKcs function in SJ formation. DNA-PKcs and the ataxia telangiectasia-mutated (ATM) kinase are members of the same family, and they share common substrates in the DNA damage response. Although ATM deficiency compromises chromosomal V(D)J CJ formation, it has no reported role in SJ formation in normal cells. Here, we report that DNA-PKcs and ATM have redundant functions in SJ formation. Thus, combined DNA-PKcs and ATM deficiency during V(D)J recombination leads to accumulation of unjoined SEs and lack of SJ fidelity. Moreover, treatment of DNA-PKcs- or ATM-deficient cells, respectively, with specific kinase inhibitors for ATM or DNA-PKcs recapitulates SJ defects, indicating that the overlapping V(D)J recombination functions of ATM and DNA-PKcs are mediated through their kinase activities. [ABSTRACT FROM AUTHOR]

Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process. [ABSTRACT FROM AUTHOR]

Upon DNA damage, histone H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other phosphoinositide 3-kinase-related protein kinases. To elucidate further the potential overlapping and unique functions of ATM and H2AX, we asked whether they have synergistic functions in the development and maintenance of genomic stability by inactivating both genes in mouse germ line. Combined ATM/H2AX deficiency caused embryonic lethality and dramatic cellular genomic instability. Mechanistically, severe genomic instability in the double-deficient cells is associated with a requirement for H2AX to repair oxidative DNA damage resulting from ATM deficiency. We discuss these findings in the context of synergies between ATM and other repair factors. [ABSTRACT FROM AUTHOR]

Nonhomologous DNA end-joining (NHEJ) is a major pathway of DNA double-strand break (DSB) repair in mammalian cells, and it functions to join both specifically programmed DSBs that occur in the context of V(D)J recombination during early lymphocyte development as well as general DSBs that occur in all cells. Thus, defects in NHEJ impair V(D)J recombination and lead to general genomic instability. In human patients, mutations of Cernunnos-XLF (also called NHEJ1), a recently identified NHEJ factor, underlie certain severe combined immune deficiencies associated with defective V(D)J recombination and radiosensitivity. To characterize Cernunnos-XLF function in mouse cells, we used gene-targeted mutation to delete exons 4 and 5 from both copies of the Cernunnos-XLF gene in ES cell (referred to as CerΔ/Δ ES cells). Analyses of CerΔ/Δ ES cells showed that they produce no readily detectable Cernunnos-XLF protein. Based on transient V(D)J recombination assays, we find that CerΔ/Δ ES cells have dramatic impairments in ability to form both V(D)J coding joins and joins of their flanking recombination signal sequences (RS joins). CerΔ/Δ ES cells are highly sensitive to ionizing radiation and have intrinsic DNA DSB repair defects as measured by pulse field gel electrophoresis. Finally, the Cernunnos-XLF mutations led to increased spontaneous genomic instability, including translocations. We conclude that, in mice, Cernunnos-XLF is essential for normal NHEJ-mediated repair of DNA DSBs and that Cernunnos-XLF acts as a genomic caretaker to prevent genomic instability. [ABSTRACT FROM AUTHOR]

Stem respiration in 20-year-old Scots pine (Pinus sylvestris L.) trees was examined following 5 years of exposure to ambient conditions (CON), elevated atmospheric carbon dioxide concentration ([CO2]) (ambient + 350 µmol mol–1, (EC)), elevated temperature (ambient + 2–6 °C, (ET)) or a combination of elevated [CO2] and elevated temperature (ECT). Stem respiration varied seasonally regardless of the treatment and displayed a similar trend to temperature, with maximum rates occurring around Day 190 in summer and minimum rates in winter. Respiration normalized to 15 °C (R 15) was higher in the growing season than in the non-growing season, whereas the temperature coefficient (Q 10) was lower in the growing season. Annually averaged R 15 was 0.36, 0.43, 0.40 and 0.44 µmol m–2 s–1 under CON, EC, ET and ECT conditions, respectively, whereas the corresponding values for total stem respiration were 6.55, 7.69, 7.50 and 7.90 mol m–2 year–1. The EC, ET and ECT treatments increased R 15 by 18, 11 and 22%, respectively, relative to CON, and increased the modeled annual total stem respiration by 18, 15 and 21%. The increase in modeled annual stem respiration under EC and ECT conditions was caused mainly by higher maintenance respiration (22 and 25%, respectively, whereas the increase in growth respiration was 9 and 12%). Growth respiration was unaltered by ET. The treatments did not significantly affect the respiratory response to stem temperature; the mean Q 10 value was 2.04, 2.10, 1.99 and 2.12 in the CON, EC, ET and ECT treatments, respectively. It is suggested that the increase in stem respiration was partly a result of the increased growth rate. We conclude that elevated [CO2] increased the maintenance component of respiration more than the growth component. [ABSTRACT FROM AUTHOR]

The article focuses on the evaluation of the role of Mre11 proteins in DNA repair beyond homologous recombination particularly in mammals. It presents homologous recombination as the one that repairs postreplicative DSBs through the use of an intact template from a sister chromatid. It documents the role of Mre11 in non-homologous end joining (NHEJ) among mammals. However, the study also reveals that the specific roles of NHEJ remain to be sort out.