DNA resection in eukaryotes: deciding how to fix the break

DNA double-strand breaks are repaired by different mechanisms, including homologous recombination and nonhomologous end-joining. DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease. Here, I review recent findings on the mechanisms of resection in eukaryotes, from yeast to vertebrates, provide insights into the regulatory strategies that control it, and highlight the consequences of both its impairment and its deregulation

Human RAD18 Interacts with Ubiquitylated Chromatin Components and Facilitates RAD9 Recruitment to DNA Double Strand Breaks

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2

Understanding the limitations of radiation-induced cell cycle checkpoints

The DNA damage response pathways involve processes of double-strand break (DSB) repair and cell cycle checkpoint control to prevent or limit entry into S phase or mitosis in the presence of unrepaired damage. Checkpoints can function to permanently remove damaged cells from the actively proliferating population but can also halt the cell cycle temporarily to provide time for the repair of DSBs. Although efficient in their ability to limit genomic instability, checkpoints are not foolproof but carry inherent limitations. Recent work has demonstrated that the G1/S checkpoint is slowly activated and allows cells to enter S phase in the presence of unrepaired DSBs for about 4–6 h post irradiation. During this time, only a slowing but not abolition of S-phase entry is observed. The G2/M checkpoint, in contrast, is quickly activated but only responds to a level of 10–20 DSBs such that cells with a low number of DSBs do not initiate the checkpoint or terminate arrest before repair is complete. Here, we discuss the limitations of these checkpoints in the context of the current knowledge of the factors involved. We suggest that the time needed to fully activate G1/S arrest reflects the existence of a restriction point in G1-phase progression. This point has previously been defined as the point when mitogen starvation fails to prevent cells from entering S phase. However, cells that passed the restriction point can respond to DSBs, albeit with reduced efficiency

Inhibition of immunoglobulin E synthesis through FcγRII (CD32) by a mechanism independent of B-cell receptor co-cross-linking

The inhibitory effect on antibody production by immune complexes has been shown to depend on co-ligation of the B-cell antigen receptor (BCR) with the low-affinity receptor for immunoglobulin G (IgG) (FcγRIIb, CD32). Here we report that immunoglobulin E (IgE) synthesis, induced in a BCR-independent manner by interleukin-4 (IL-4) and anti-CD40 antibody, was inhibited by CD32 ligation. The observed effect was specific for CD32 as, first, antibodies directed against other B-cell surface structures had no inhibitory effect, and, second, treatment with anti-CD32 of cells that had been in culture for 2 days was ineffective owing to the down-regulation of CD32 expression. IgE inhibition was also observed in cells stimulated by IL-4/CD40 F(ab′)(2) or IL-4 plus soluble CD40 ligand, demonstrating that co-cross-linking of CD32 and CD40 was not necessary to induce inhibition. Mechanistic studies into the IgE class switch process demonstrated that IL-4/anti-CD40-induced IgE germline gene transcription and B-cell proliferation were not affected by CD32 ligation. The data demonstrate that the negative regulatory role of the CD32 molecule is not restricted to BCR-induced B-cell activation, but is also functional on other B-cell activation pathways mediated by CD40 and IL-4

Copy number variation of Fc gamma receptor genes in HIV-infected and HIV-tuberculosis co-infected individuals in Sub-Saharan Africa

AIDS, caused by the retrovirus HIV, remains the largest cause of morbidity in sub-Saharan Africa yet almost all genetic studies have focused on cohorts from Western countries. HIV shows high co-morbidity with tuberculosis (TB), as HIV stimulates the reactivation of latent tuberculosis (TB). Recent clinical trials suggest that an effective anti-HIV response correlates with non-neutralising antibodies. Given that Fcγ receptors are critical in mediating the nonneutralising effects of antibodies, analysis of the extensive variation at Fcγ receptor genes is important. Single nucleotide variation and copy number variation (CNV) of Fcγ receptor genes affects the expression profile, activatory/inhibitory balance, and IgG affinity of the Fcγ receptor repertoire of each individual. In this study we investigated whether CNV of FCGR2C, FCGR3A and FCGR3B as well as the HNA1 allotype of FCGR3B is associated with HIV load, response to highly-active antiretroviral therapy (HAART) and co-infection with TB. We confirmed an effect of TB-co-infection status on HIV load and response to HAART, but no conclusive effect of the genetic variants we tested. We observed a small effect, in Ethiopians, of FCGR3B copy number, where deletion was more frequent in HIV-TB co-infected patients than those infected with HIV alone