DNA ligase IV mutations confer shorter lifespan and increased sensitivity to nutrient stress in Drosophila melanogaster

The nonhomologous end-joining pathway is a primary DNA double-strand break repair pathway in eukaryotes. DNA ligase IV (Lig4) catalyzes the final step of DNA end ligation in this pathway. Partial loss of Lig4 in mammals causes Lig4 syndrome, while complete loss is embryonically lethal. DNA ligase 4 (DNAlig4) null Drosophila melanogaster is viable, but sensitive to ionizing radiation during early development. We proposed to explore if DNAlig4 loss induced other long-term sensitivities and defects in D. melanogaster. We demonstrated that DNAlig4 mutant strains had decreased lifespan and lower resistance to nutrient deprivation, indicating Lig4 is required for maintaining health and longevity in D. melanogaster.

CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

Characterization of a Cohort of Patients With LIG4 Deficiency Reveals the Founder Effect of p.R278L, Unique to the Chinese Population

DNA ligase IV (LIG4) deficiency is an extremely rare autosomal recessive primary immunodeficiency disease caused by mutations in LIG4. Patients suffer from a broad spectrum of clinical problems, including microcephaly, growth retardation, developmental delay, dysmorphic facial features, combined immunodeficiency, and a predisposition to autoimmune diseases and malignancy. In this study, the clinical, molecular, and immunological characteristics of 15 Chinese patients with LIG4 deficiency are summarized in detail. p.R278L (c.833G>T) is a unique mutation site present in the majority of Chinese cases. We conducted pedigree and haplotype analyses to examine the founder effect of this mutation site in China. This suggests that implementation of protocols for genetic diagnosis and for genetic counseling of affected pedigrees is essential. Also, the search might help determine the migration pathways of populations with Asian ancestry.

A XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development

We developed a Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4 deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA Ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core NHEJ DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double KO settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights in the understanding of the clinical manifestations of human XRCC4 deficient condition, in particular its absence of immune deficiency.

Haploidentical Transplantation in a DNA Ligase IV Deficiency Patient: A Case Report and Review of The Literature

Background: DNA ligase IV (LIG4) deficiency is a rare autosomal recessive disorder caused by mutations in the DNA LIG4 gene. Nowadays hematopoietic stem cell transplantation(HSCT) is the most effective treatment option. Matched sibling or unrelated donor was the best choice for those patients. However, it will be a problem for LIG4 deficiency patients without proper donor as above but needed urgent transplantation because of infection or bone marrow failure. Furthermore, mixed donor chimerism after transplantation is more common in LIG4 deficiency patients because of reduced intensity conditioning(RIC) regimen. How to deal with those problems? Here we report a case which the patient received haploidentical HSCT and got complete donor chimerism after donor stem cell infusion. Results:The patient was diagnosed as LIG4 deficiency at 8-month-old. At 14 months of age, she received a T cell receptor(TCR)α/β and CD19+ B cells depleted graft from his haploidentical father, followed a RIC regimen with no additional graft versus host disease(GvHD) prophylaxis. Engraftment was as usual. However, mixed donor chimerism occurred after transplantation and viremia persisted. Cryopreserved donor cell infusion was initiated. The chimerism grew up steadily and viremia disappeared at four months post transplantation. Conclusions: This case report gives an example of successful haploidentical transplantation and donor stem cell infusion as a treatment option in a mixed donor chimerism situation after HSCT in LIG4 deficiency patients.

Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells

Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but predominates in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 (“Ku”) and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Ligase IV are absolutely required for joining RAG1/2-endonucease (“RAG”)-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Ligase IV also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Ligase IV-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially, in association with G1-phase down-regulation of Ligase1. Finally, we suggest that differential impacts of Ku-deficiency versus Lig4-deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-defcient developing lymphocyte and neuronal cells.Significance StatementAlternative end-joining (A-EJ) is implicated in oncogenic translocations and mediating DNA double-strand break (DSB) repair in cycling cells when classical nonhomologous endjoining (C-NHEJ) factors of the C-NHEJ Ligase complex are absent. However, V(D)J recombination-associated DSBs that occur in G1 cell cycle-phase progenitor lymphocytes are joined exclusively by the C-NHEJ pathway. Until now, however, the overall mechanisms that join general DSBs in G1-phase progenitor B cells had not been fully elucidated. Here, we report that Ku, a core C-NHEJ double-strand break recognition complex, directs repair of a variety of different targeted DSBs towards C-NHEJ and suppresses A-EJ in G1-phase cells. We suggest this Ku activity explains how Ku-deficiency can rescue the neuronal development and embryonic lethality phenotype of Ligase 4-deficient mice.

NAD+ is not utilized as a co-factor for DNA ligation by human DNA ligase IV

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.