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.

DNA polymerase delta, whose catalytic subunit is encoded by POLD1, is responsible for lagging strand DNA synthesis during DNA replication1. It achieves this with high fidelity due to its intrinsic 3′ to 5′ exonuclease activity, which confers proofreading ability. Missense mutations in the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancer2. Here we report a recurring heterozygous single amino acid deletion at the polymerase active site of POLD1 that abolishes DNA polymerase activity but only mildly impairs 3′ to 5′ exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This suggests that perturbation of function of the ubiquitously expressed POLD1 polymerase has surprisingly tissue-specific effects in man, and argues for an important role for POLD1 function in adipose tissue homeostasis.

The detection of minority variants in mixed samples demands methods for enrichment and accurate sequencing of small genomic intervals. We describe an efficient approach based on sequential rounds of hybridization with biotinylated oligonucleotides, enabling more than one-million fold enrichment of genomic regions of interest. In conjunction with error correcting double-stranded molecular tags, our approach enables the quantification of mutations in individual DNA molecules.

The eukaryotic genome is in a constant state of modification and repair. Faithful transmission of the genomic information from parent to daughter cells depends upon an extensive system of surveillance, signaling, and DNA repair, as well as accurate synthesis of DNA during replication. Often, replicative synthesis occurs over regions of DNA that have not yet been repaired, presenting further challenges to genomic stability. DNA polymerase δ (Pol δ) occupies a central role in all of these processes: catalyzing the accurate replication of a majority of the genome, participating in several DNA repair synthetic pathways, and contributing structurally to the accurate bypass of problematic lesions during translesion synthesis. The concerted actions of pol δ on the lagging strand, pol ε on the leading strand, associated replicative factors, and the mismatch repair (MMR) proteins results in a mutation rate of less than one misincorporation per genome per replication cycle. This low mutation rate provides a high level of protection against genetic defects during development and may prevent the initiation of malignancies in somatic cells. This review explores the role of Pol δ in replication fidelity and genome maintenance.

The Pax transactivation domain-interacting protein (PTIP) is a large nuclear protein with multiple BRCT domains that was identified on the basis of its interaction with transcription factors of the Pax and Smad families. To address the function of PTIP during mouse development, we generated a constitutive null allele. Homozygous PTIP mutants are developmentally retarded, disorganized, and embryonic lethal by day 9.5 of embryonic development (E9.5). PTIP mutant cells appear to replicate DNA but show reduced levels of mitosis and widespread cell death by E8.5. DNA damage appears to precede nuclear condensation at E7.5, suggesting a defect in DNA repair. Neither embryonic fibroblast nor embryonic stem cells from PTIP mutants proliferate in culture, suggesting a fundamental defect in cell proliferation. Trophoblast cells from PTIP mutants are more sensitive to DNA-damaging agents. Condensation of chromatin and expression of phospho-histone H3 are also affected in PTIP mutants, and this may underlie the inability of PTIP mutants to progress through mitosis. Given the role of BRCT domain proteins in DNA repair and cell cycle control, we propose that PTIP is an essential element of the cell proliferation machinery, perhaps by functioning in the DNA repair pathways.Murine PTIP (Pax transactivation domain-interacting protein) was identified on the basis of its interaction with the Pax family of DNA binding transcription factors (13). PTIP is a large nuclear protein with multiple BRCT domains. The BRCT domain was originally described in the carboxy terminus of the BRCA1 tumor suppressor protein (12), yet hydrophobic cluster analysis revealed a large family of nuclear proteins, across divergent species, with similar BRCT repeats. Like BRCA1, many of these genes are thought to function in regulation of the cell cycle, in activation of cell cycle checkpoints, and/or in mediation of responses to DNA-damaging agents. For example, BRCA1 activates the G 2 /M checkpoint, in response to DNA damage, through the Chk1 kinase (24). The BRCT domains are essential for BRCA1 activity, as both human mutations and targeted mouse mutations have demonstrated (8). In yeast, the BRCT domain protein Cut5/Rad4 interacts with a second BRCT domain protein, Crb2, to activate the DNA damage checkpoint (18). Phosphorylation of Crb2 by cdc2/CDK1 is necessary for re-entry into mitosis after DNA repair (5) and for topoisomerase III function in recombination-mediated repair (2).In addition to their potential roles in DNA repair pathways, the nuclear BRCT domain proteins have also been linked to gene activation (22). In Xenopus laevis, the PTIP-related protein Swift was identified on the basis of its ability to bind the Smad2 transcription factor and accentuate Smad2-dependent transcription (20). At the time of gastrulation, Smad2 mediates gene activation in response to signaling by the secreted transforming growth factor ␤ family member Activin. PTIP also interacts with the Pax family of transcription regulators (13) and, at least in one case, is able to...

The Pax family of genes encodes transcription factors with conserved DNA binding motifs that are required for embryonic development of a variety of tissues in Drosophila, mouse, and humans. In vertebrates, Pax genes can be subdivided into classes based on similar features and embryonic expression patterns (1). The Pax2/5/8 subfamily is characterized by an amino-terminal-paired domain, a conserved octapeptide sequence with similarity to the engrailed homology domain (EH-1), and a partial-paired type homeobox. Pax2/5/8 proteins are transcription regulators that bind DNA via the amino-terminal-paired domain, whereas the carboxyl-terminal region is required for transactivation of target genes (2, 3).In mouse and man, the Pax2 gene is essential for the development of the kidneys (4), optic cup, and inner ear (5). Pax2 encodes at least two alternatively spliced messages that produce proteins of 392 and 415 amino acids, differing only by a 23 amino acid insertion (6). Genes known to be up-regulated by Pax2 include WT1 (7) and gdnf (8) in the developing kidney and engrailed-2 (9) in the developing hindbrain. In transfected cells, transcription activation requires the Pax2 carboxyl-terminal domain that is rich in serine and threonine residues, which may be potential sites for phosphorylation. In the zebrafish Pax6 protein, the carboxyl-terminal transactivation domain is phosphorylated at multiple serine residues by the mitogen-activated protein kinases (MAPK) 1 p38 MAPK and ERK1/2 to increase the transactivation potential (10). Of the Pax2/5/8 subfamily, only Pax8 has been studied with respect to phosphorylation, though it is not clear which kinases are involved (11). MAP kinase cascades are involved in transmitting signals generated at the cell surface into the cytosol and nucleus and consist of three sequentially acting enzymes: a MAP kinase, an upstream MAP kinase kinase (MEK), and a MEK kinase (MEKK) (12, 13). The extracellular signal-regulated kinase 1/2 (ERK 1/2), the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPK), or the p38MAP kinases, can translocate to the nucleus and subsequentely phosphorylate a variety of transcription factors. MEKK1 phosphorylates MKK4/MKK7 to activate JNK but can also activate ERK and p38 MAPK in transfected cells.This report addresses the phosphorylation state of the Pax2 protein and its ability to activate transcription. We show that the carboxyl-terminal activation domain is phosphorylated at serine and threonine residues and that Pax2 phosphorylation is coincident with the enhanced ability to transactivate a reporter gene. Pax2 is a substrate for the c-Jun N-terminal kinase (JNK) but, unlike Pax6, is not phosphorylated by ERK or p38 MAPK. Activation of JNK by either the upstream kinases MEKK1 or DLK or by expression of Wnt signaling proteins increases Pax2 phosphorylation and enhances the Pax2 transactivation potential. The data point to an important role for JNK in modifying the Pax2 transactivation domain and stimulating Pax2-dependent gene expression. MATERIALS ...

The mutator phenotype hypothesis proposes that the mutation rate of normal cells is insufficient to account for the large number of mutations found in human cancers. Consequently, human tumors exhibit an elevated mutation rate that increases the likelihood of a tumor acquiring advantageous mutations. The hypothesis predicts that tumors are composed of cells harboring hundreds of thousands of mutations, as opposed to a small number of specific driver mutations, and that malignant cells within a tumor therefore constitute a highly heterogeneous population. As a result, drugs targeting specific mutated driver genes or even pathways of mutated driver genes will have only limited anticancer potential. In addition, because the tumor is composed of such a diverse cell population, tumor cells harboring drug-resistant mutations will exist prior to the administration of any chemotherapeutic agent. We present recent evidence in support of the mutator phenotype hypothesis, major arguments against this concept, and discuss the clinical consequences of tumor evolution fueled by an elevated mutation rate. We also consider the therapeutic possibility of altering the rate of mutation accumulation. Most significantly, we contend that there is a need to fundamentally reconsider current approaches to personalized cancer therapy. We propose that targeting cellular pathways that alter the rate of mutation accumulation in tumors will ultimately prove more effective than attempting to identify and target mutant driver genes or driver pathways.

DNA sequencing studies have established that many cancers contain tens of thousands of clonal mutations throughout their genomes, a fact which is difficult to reconcile with the very low rate of mutation in normal human cells. This observation provides strong evidence for the mutator phenotype hypothesis, which proposes that an elevation in the spontaneous mutation rate is an early step in carcinogenesis. An elevated mutation rate implies that cancers undergo continuous evolution and harbor multiple sub-populations of cells differing from one another in DNA sequence. The extensive heterogeneity in DNA sequence and continual tumor evolution that would occur in the context of a mutator phenotype have important implications for cancer diagnosis and therapy.