EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart.

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.

Role of HP1ß during spermatogenesis and DNA replication.

Heterochromatin protein 1ß (HP1ß), encoded by the Cbx1 gene, has been functionally linked to chromatin condensation, transcriptional regulation, and DNA damage repair. Here we report that testis-specific Cbx1 conditional knockout (Cbx1 cKO) impairs male germ cell development in mice. Depletion of HP1ß negatively affected sperm maturation and increased seminiferous tubule degeneration in Cbx1 cKO mice. In addition, the spermatogonia have elevated γ-H2AX foci levels as do Cbx1 deficient mouse embryonic fibroblasts (MEFs) as compared to wild-type (WT) control MEFs. The increase in γ-H2AX foci in proliferating Cbx1 cKO cells indicates defective replication-dependent DNA damage repair. Depletion or loss of HP1ß from human cells and MEFs increased DNA replication fork stalling and firing of new origins of replication, indicating defective DNA synthesis. Taken together, these results suggest that loss of HP1ß in proliferating cells leads to DNA replication defects with associated DNA damage that impact spermatogenesis.

Functional characterization of the role of the chromosome I partitioning system in genome segregation in Deinococcus radiodurans.

Deinococcus radiodurans, a radiation-resistant bacterium, harbors a multipartite genome. Chromosome I contains three putative centromeres (segS1, segS2, and segS3), and ParA (ParA1) and ParB (ParB1) homologues. The ParB1 interaction with segS was sequence specific, and ParA1 was shown to be a DNA binding ATPase. The ATPase activity of ParA1 was stimulated when segS elements were coincubated with ParB1, but the greatest increase was observed with segS3. ParA1 incubated with the segS-ParB1 complex showed increased light scattering in the absence of ATP. In the presence of ATP, this increase was continued with segS1-ParA1B1 and segS2-ParA1B1 complexes, while it decreased rapidly after an initial increase for 30 min in the case of segS3. D. radiodurans cells expressing green fluorescent protein (GFP)-ParB1 produced foci on nucleoids, and the ΔparB1 mutant showed growth retardation and ∼13%-higher anucleation than the wild type. Unstable mini-F plasmids carrying segS1 and segS2 showed inheritance in Escherichia coli without ParA1B1, while segS3-mediated plasmid stability required the in trans expression of ParA1B1. Unlike untransformed E. coli cells, cells harboring pDAGS3, a plasmid carrying segS3 and also expressing ParB1-GFP, produced discrete GFP foci on nucleoids. These findings suggested that both segS elements and the ParA1B1 proteins of D. radiodurans are functionally active and have a role in genome segregation.

Caspase-2 regulates S-phase cell cycle events to protect from DNA damage accumulation independent of apoptosis.

In addition to its classical role in apoptosis, accumulating evidence suggests that caspase-2 has non-apoptotic functions, including regulation of cell division. Loss of caspase-2 is known to increase proliferation rates but how caspase-2 is regulating this process is currently unclear. We show that caspase-2 is activated in dividing cells in G1-phase of the cell cycle. In the absence of caspase-2, cells exhibit numerous S-phase defects including delayed exit from S-phase, defects in repair of chromosomal aberrations during S-phase, and increased DNA damage following S-phase arrest. In addition, caspase-2-deficient cells have a higher frequency of stalled replication forks, decreased DNA fiber length, and impeded progression of DNA replication tracts. This indicates that caspase-2 protects from replication stress and promotes replication fork protection to maintain genomic stability. These functions are independent of the pro-apoptotic function of caspase-2 because blocking caspase-2-induced cell death had no effect on cell division, DNA damage-induced cell cycle arrest, or DNA damage. Thus, our data supports a model where caspase-2 regulates cell cycle and DNA repair events to protect from the accumulation of DNA damage independently of its pro-apoptotic function.

Autism-Associated Vigilin Depletion Impairs DNA Damage Repair.

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, and mRNA stability and is associated with autism spectrum disorders and cancer: vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains. Here, we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX and elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 focus formation, DNA end resection, and double-strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU)-induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in the transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer-related disorders.

Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination.

The nitric oxide (NO)-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR) to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR) repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1). Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18), which causes stem cell differentiation has no effect on double-strand break (DSB) repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.