SummaryThe chromatin remodeling factor SMARCAD1, an SWI/SNF ATPase family member, has a role in 5′ end resection at DNA double-strand breaks (DSBs) to produce single-strand DNA (ssDNA), a critical step for subsequent checkpoint and repair factor loading to remove DNA damage. However, the mechanistic details of SMARCAD1 coupling to the DNA damage response and repair pathways remains unknown. Here we report that SMARCAD1 is recruited to DNA DSBs through an ATM-dependent process. Depletion of SMARCAD1 reduces ionizing radiation (IR)-induced repairosome foci formation and DSB repair by homologous recombination (HR). IR induces SMARCAD1 phosphorylation at a conserved T906 by ATM kinase, a modification essential for SMARCAD1 recruitment to DSBs. Interestingly, T906 phosphorylation is also important for SMARCAD1 ubiquitination by RING1 at K905. Both these post-translational modifications are critical for regulating the role of SMARCAD1 in DNA end resection, HR-mediated repair, and cell survival after DNA damage.

Myeloid cell leukemia 1 (MCL-1) is a prosurvival BCL-2 protein family member highly expressed in hematopoietic stem cells (HSCs) and regulated by growth factor signals that manifest antiapoptotic activity. Here we report that depletion of MCL-1 but not its isoform MCL-1S increases genomic instability and cell sensitivity to ionizing radiation (IR)-induced death. MCL-1 association with genomic DNA increased postirradiation, and the protein colocalized with 53BP1 foci. Postirradiation, MCL-1-depleted cells exhibited decreased ␥-H2AX foci, decreased phosphorylation of ATR, and higher levels of residual 53BP1 and RIF1 foci, suggesting that DNA double-strand break (DSB) repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1-depleted cells had a reduced frequency of IRinduced BRCA1, RPA, and Rad51 focus formation, decreased DNA end resection, and decreased HR repair in the DR-GFP DSB repair model. Similarly, after HU induction of stalled replication forks in MCL-1-depleted cells, there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HRmediated resolution of collapsed forks. Therefore, the present data support a model whereby MCL-1 depletion increases 53BP1 and RIF1 colocalization at DSBs, which inhibits BRCA1 recruitment, and sensitizes cells to DSBs from IR or stalled replication forks that require HR for repair.KEYWORDS MCL-1, BCL-2, HR, ICL, apoptosis, 53BP1, DSB repair M CL-1 is a member of the prosurvival BCL-2 family and plays an important role in the regulation of the intrinsic or mitochondrial apoptotic pathway by inhibiting both BH3-only proteins and the proapoptotic proteins (1-3). MCL-1 is mainly located at the outer mitochondrial membrane and inhibits the progression of apoptosis by sequestering executioner proapoptotic proteins BAK and BAX, which are capable of inducing pore formation in the mitochondrial membrane. The subsequent release of cytochrome c into the cytoplasm activates caspases which are responsible for the majority of the macromolecular degradation observed during apoptosis (3). Suppression of BAK and BAX polymerization by MCL-1 is prevented either by MCL-1 degradation or by saturating and inhibiting the MCL-1 binding sites on BAK/BAX with BH3 proteins or mimetics.Under normal growth conditions, MCL-1 is important for mouse embryonic survival (4) and critical for the survival of neutrophils, lymphocytes, hematopoietic stem cells, and neurons (5). MCL-1 overexpression is the hallmark of several cancers, including hematological malignancies as well as solid tumors. Elevated cellular MCL-1 expression correlates with resistance to drug toxicity and ionizing radiation (IR), whereas its inhibition sensitizes cells to both. The BCL-2 family of proteins is characterized by the presence of BCL-2 homology (BH) domains (1, 2). The MCL-1 protein itself is unique among BCL-2 members in also containing multiple N-terminal PEST motifs in addition to BH1, BH2, BH3, and C-terminal transmembrane (TM) domains. PEST is ...

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45 degrees C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45 degrees C by wet heat, 2 M HCl, 2 M NaOH and 2 M H(2)O(2) was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45 degrees C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45 degrees C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45 degrees C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.