Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 107 wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence ‘families’ that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1–3 d.

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.

ING2 is a candidate tumor suppressor gene that can activate p53 by enhancing its acetylation. Here, we demonstrate that ING2 is also involved in p53-mediated replicative senescence. ING2 protein expression increased in late-passage human primary cells, and it colocalizes with serine 15-phosphorylated p53. ING2 and p53 also complexed with the histone acetyltransferase p300. ING2 enhanced the interaction between p53 and p300 and acted as a cofactor for p300-mediated p53 acetylation. The level of ING2 expression directly modulated the onset of replicative senescence. While overexpression of ING2 induced senescence in young fibroblasts in a p53-dependent manner, expression of ING2 small interfering RNA delayed the onset of senescence. Hence, ING2 can act as a cofactor of p300 for p53 acetylation and thereby plays a positive regulatory role during p53-mediated replicative senescence.

The WRN DNA helicase is a member of the DExH-containing DNA helicase superfamily that includes XPB, XPD, and BLM. Mutations in WRN are found in patients with the premature aging and cancer susceptibility syndrome known as Werner syndrome (WS). p53 binds to the WRN protein in vivo and in vitro through its carboxyl terminus. WS fibroblasts have an attenuated p53-mediated apoptotic response, and this deficiency can be rescued by expression of wild-type WRN. These data support the hypothesis that p53 can induce apoptosis through the modulation of specific DExH-containing DNA helicases and may have implications for the cancer predisposition observed in WS patients.

The ING4 (inhibitor of growth family member 4) was identified in our laboratory as a candidate tumor suppressor gene (1) that is down-regulated in glioblastoma cells (2) and head and neck squamous cell carcinoma (3). ING4 suppresses cell growth (1, 4, 5), suppresses the loss of contact inhibition (6), inhibits angiogenesis (2), and down-regulates the stability of hypoxiainducible factor (HIF) 3 -␣ (7) through a physical interaction with HIF prolyl hydroxylase (HPH)-2C (8), resulting in repressed HIF activation in a chromatin-dependent manner. More recently, it was reported that ING4 acetylates histone H4 lysine 5, 8, and 12 with a histone acetyltransferase, HBO1/ MYST2 (MYST2 is MOZ, YBF2/SAS3, SAS2 and TIP60 histone acetyltransferase 2) (9), and interacts with methylated histone H3 (10). Thus, ING4 seems to play several major roles in cells like other ING family proteins (11) that are conserved from yeasts to vertebrates (12).In this study, we describe three novel splice variants of ING4, ING4_v2 (a 3-bp skip form), ING4_v3 (a 9-bp skip form), and ING4_v4 (a 12-bp skip form), besides ING4_v1 (the originally enrolled ING4, the longest form). These variants are produced from the alternative use of two splice donor sites at the end of exon 4 and two splice acceptor sites at the start of exon 5 of the ING4 gene, although one of the three variants, ING4_v4, was previously attributed to a common deletion mutation (6). The alternative RNA splicing of the ING4 pre-mRNA causes a partial loss of an NLS and affects nuclear localization of the proteins. We show that the small deletion in the ING4 protein leads to functional differences between the ING4 variants. Growth suppressive effects of the variants that have the partially missing NLS were attenuated by weaker activity on p21 WAF1 induction. In addition, ING4_v4 showed attenuated suppressive effects on cell spreading and migration compared with the original ING4 (ING4_v1). On the other hand, ING4_v2 only lost the suppressive effect on cell spreading, suggesting an important role for ING4_v2 on the regulation of cell migration. ING4_v4 played dominant-negative roles on these ING4_v1 effects. In addition, we found that ING4_v1 and ING4_v2, but not ING4_v4, have a binding affinity to Liprin ␣1 that may play a role in the regulation of focal adhesion disassembly (13,14). In addition, only ING_v1 has a binding affinity to G3BP2a, which is involved in Ras signaling, NF-B signaling, and the ubiquitin proteasome system (15-17). These differences may affect the function of ING4 splice variants. 3 The abbreviations used are: HIF, hypoxia-inducible factor; NLS, nuclear localization signal; Liprin ␣1, LAR-interacting protein ␣1; G3BP2, RasGTPase activating protein SH3 domain-binding protein 2; PHD, plant homeodomain; NF-B, nuclear factor of B; HPH-2, HIF prolyl hydroxylase 2; ESE, exonic splicing enhancer.

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Inhibitor of growth 4 (ING4) is a candidate tumor suppressor that plays a major role in gene regulation, cell cycle control, apoptosis, and angiogenesis. ING4 expression is downregulated in glioblastoma cells and head and neck squamous cell carcinoma. Here, we identified liprin A1/PPFIA1, a cytoplasmic protein necessary for focal adhesion formation and axon guidance, as a novel interacting protein with ING4. ING4 and liprin A1 colocalized at lamellipodia in the vicinity of vinculin. Overexpressed ING4 suppressed cell spreading and cell migration. In contrast, overexpressed liprin A1 enhanced cell spreading and cell migration. Knockdown of endogenous ING4 with RNA interference induced cell motility, whereas knockdown of endogenous liprin A1 suppressed cell motility. ING4 also suppressed cell motility that was enhanced by liprin A1. However, ING4 did not further suppress cell motility when liprin A1 was suppressed with RNA interference, suggesting a functional and mechanistic interdependence between these proteins. In addition to its nuclear functions, cytoplasmic ING4 interacts with liprin A1 to regulate cell migration and, with its known antiangiogenic function, may prevent invasion and metastasis. [Cancer Res 2007;67(6):2552-8]