Eric A. Hendrickson

Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.

A sequential two-step mechanism for the production of the mature p17: p12 form of caspase-3 in vitro

The apoptotic cysteine protease, caspase-3, is expressed in cells as an inactive 32-kDa precursor from which 17 kDa (p17) and 12 kDa (p12) subunits of the mature caspase-3 are proteolytically generated during apoptosis. Two amino acid sequences, ESMD↓S (amino acids 25–29) and IETD↓S (amino acids 172–176), in the precursor have been defined as the cleavage sites for the production of the p17 and p12 subunits. Using a cell-free assay system, we demonstrate that the caspase-3 precursor appears to be cleaved first at the IETD↓S site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD↓S site, generating the mature p17 subunit. The cleavage at the IETD↓S site required a protease activity that was selectively inhibited by the peptide, Ac-IETD-CHO (acetyl-IETD-aldehyde), and other protease inhibitors, such as the cowpox viral serine protease inhibitor, CrmA, and N-α-tosyl-l-phenylalanine chloromethyl ketone. The protease that catalyzed the cleavage at the ESMD/S site was selectively inhibited by another peptide, Ac-ESMD-CHO (acetyl-ESMD-aldehyde). More interestingly, the caspase-3 inhibitor, Ac-DEVD-CHO, but not the caspase-1 inhibitor, Ac-YVAD-CHO, also selectively inhibited the protease activity that cleaves at the ESMD↓S site. This indicated that the cleavage at the ESMD↓S site was either autocatalytic or that it required a caspase-3-like activity. In summary, we demonstrate that production of the p17:p12 form of caspase-3 is a sequential two-step process and appears to require two distinct enzymatic activities.

Role of p21 in Apoptosis and Senescence of Human Colon Cancer Cells Treated with Camptothecin

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53−/−HCT116 and p21−/− HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53−/− HCT116 and p21−/− HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21−/− HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

Ku86 is essential in human somatic cells

Ku86 plays a key role in nonhomologous end joining in mammals. Functional inactivation in rodents of either Ku86 or Ku70, which form the heterodimeric DNA end-binding subunit of the DNA-dependent protein kinase complex, is nevertheless compatible with viability. In contrast, no human patient has been described with mutations in either Ku86 or Ku70. This has led to the hypotheses that either these genes are performing an additional essential role(s) and/or redundant pathways exist that mask the phenotypic expression of these genes when they are mutated in humans. To address this issue, we describe here the construction of human somatic cell lines containing a targeted disruption of the Ku86 locus. Human HCT116 colon cancer cells heterozygous for Ku86 were haploinsufficient with an increase in polyploid cells, a reduction in cell proliferation, elevated p53 levels, and a slight hypersensitivity to ionizing radiation. Functional inactivation of the second Ku86 allele resulted in cells with a drastically reduced doubling time. These cells were capable of undergoing only a limited number of cell divisions, after which they underwent apoptosis. These experiments demonstrate that the Ku86 locus is essential in human somatic tissue culture cells.

Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining

Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

DNA-dependent Protein Kinase Is a Target for a CPP32-like Apoptotic Protease

We demonstrate that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is specifically, proteolytically cleaved in HL-60 cells treated with staurosporine (STS), a potent inducer of apoptosis. The proteolysis of DNA-PKcs correlated with or preceded apoptotic chromosomal DNA degradation. Cell-free extracts prepared from STS-treated HL-60 cells recapitulated the proteolysis of DNA-PKcs in an in vitro assay using purified DNA-PK as the substrate. Western blot analyses of the apoptotic cell extract showed that the 32-kDa precursor of CPP32 is expressed in HL-60 cells and processed following STS treatment. In addition, whereas the DNA-PKcs protease activity was not inhibitable by many conventional protease inhibitors, it was inhibitable by a highly selective peptide-derived inhibitor of CPP32. These data strongly suggest that CPP32, or a CPP32-like protease, is responsible for DNA-PKcs proteolysis. Finally, our results demonstrated that the cleavage of DNA-PKcs in vitro proceeded in the presence of Bcl-2, indicating that the function provided by Bcl-2 lies upstream the proteolysis of DNA-PKcs.

DNA2 drives processing and restart of reversed replication forks in human cells

Following prolonged genotoxic stress, DNA2 and WRN functionally interact to degrade reversed replication forks and promote replication restart, thereby preventing aberrant processing of unresolved replication intermediates

Ku86 represses lethal telomere deletion events in human somatic cells

Nonhomologous end joining (NHEJ), a form of DNA double-strand break (DSB) repair, is conserved from bacteria to humans. One essential NHEJ factor is Ku, which consists of a heterodimer of Ku70 and Ku86. In a plethora of model systems, null mutations for Ku70 or Ku86 present with defects in DNA DSB repair, variable(diversity)joining [V(D)J] recombination, and/or telomere maintenance. The complete loss of Ku from bacteria to mice is, however, compatible with viability. In striking contrast, human patients with mutations of either Ku subunit have never been described. Here, we have used recombinant adeno-associated virus-mediated gene targeting to produce a human somatic cell line that expresses a conditionally null allele of Ku86. The induced loss of Ku86 results in cell death accompanied by massive telomere loss in the form of t-circles. Thus, Ku86 is an essential gene in human somatic cells because of its requirement, not in NHEJ or V(D)J recombination, but in telomere maintenance.

Regulation of Telomere Length and Suppression of Genomic Instability in Human Somatic Cells by Ku86

ABSTRACT Ku86 plays a key role in nonhomologous end joining in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells, or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We demonstrate here that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations, and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs the additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length.