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We analyzed 921 adenocarcinomas of the esophagus, stomach, colon, and rectum to examine shared and distinguishing molecular characteristics of gastrointestinal tract adenocarcinomas (GIACs). Hypermutated tumors were distinct regardless of cancer type and comprised those enriched for insertions/deletions, representing microsatellite instability cases with epigenetic silencing of MLH1 in the context of CpG island methylator phenotype, plus tumors with elevated single-nucleotide variants associated with mutations in POLE. Tumors with chromosomal instability were diverse, with gastroesophageal adenocarcinomas harboring fragmented genomes associated with genomic doubling and distinct mutational signatures. We identified a group of tumors in the colon and rectum lacking hypermutation and aneuploidy termed genome stable and enriched in DNA hypermethylation and mutations in KRAS, SOX9, and PCBP1.
Copyright © 2018 Elsevier Inc. All rights reserved.
Billions of base pairs of DNA must be replicated trillions of times in a human lifetime. Complete and accurate replication once and only once per cell division cycle is essential to maintain genome integrity and prevent disease. Impediments to replication fork progression including difficult to replicate DNA sequences, conflicts with transcription, and DNA damage further add to the genome maintenance challenge. These obstacles frequently cause fork stalling, but only rarely cause a failure to complete replication. Robust mechanisms ensure that stalled forks remain stable and capable of either resuming DNA synthesis or being rescued by converging forks. However, when failures do happen the fork collapses leading to genome rearrangements, cell death and disease. Despite intense interest, the mechanisms to repair damaged replication forks, stabilize them, and ensure successful replication remain only partly understood. Different models of fork collapse have been proposed with varying descriptions of what happens to the DNA and replisome. Here, I will define fork collapse and describe what is known about how the replication checkpoint prevents it to maintain genome stability.
Copyright © 2015 Elsevier B.V. All rights reserved.
The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3' to 5' exonuclease activities in vitro. Steady-state kinetic efficiencies (k(cat)/K(m)) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.
The oligodeoxynucleotide 5'-CGCATXGAATCC-3'·5'-GGATTCAATGCG-3' containing 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdG) opposite deoxyadenosine (named the 1,N(2)-εdG·dA duplex) models the mismatched adenine product associated with error-prone bypass of 1,N(2)-εdG by the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) and by Escherichia coli polymerases pol I exo(-) and pol II exo(-). At pH 5.2, the T(m) of this duplex was increased by 3 °C as compared to the duplex in which the 1,N(2)-εdG lesion is opposite dC, and it was increased by 2 °C compared to the duplex in which guanine is opposite dA (the dG·dA duplex). A strong NOE between the 1,N(2)-εdG imidazole proton and the anomeric proton of the attached deoxyribose, accompanied by strong NOEs to the minor groove A(20) H2 proton and the mismatched A(19) H2 proton from the complementary strand, establish that 1,N(2)-εdG rotated about the glycosyl bond from the anti to the syn conformation. The etheno moiety was placed into the major groove. This resulted in NOEs between the etheno protons and T(5) CH(3). A strong NOE between A(20) H2 and A(19) H2 protons established that A(19), opposite to 1,N(2)-εdG, adopted the anti conformation and was directed toward the helix. The downfield shifts of the A(19) amino protons suggested protonation of dA. Thus, the protonated 1,N(2)-εdG·dA base pair was stabilized by hydrogen bonds between 1,N(2)-εdG N1 and A(19) N1H(+) and between 1,N(2)-εdG O(9) and A(19)N(6)H. The broad imino proton resonances for the 5'- and 3'-flanking bases suggested that both neighboring base pairs were perturbed. The increased stability of the 1,N(2)-εdG·dA base pair, compared to that of the 1,N(2)-εdG·dC base pair, correlated with the mismatch adenine product observed during the bypass of 1,N(2)-εdG by the Dpo4 polymerase, suggesting that stabilization of this mismatch may be significant with regard to the biological processing of 1,N(2)-εdG.
© 2011 American Chemical Society
G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by noncanonical G-G base pairs. Genome-wide chromatin immunoprecipitation was used to determine the in vivo binding sites of the multifunctional Saccharomyces cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in vitro. G4 motifs were a significant subset of the high-confidence Pif1-binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, whereas non-G4 Pif1-binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication-stressed Pif1-deficient cells, which was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication, and stimulate DNA breakage. These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage.
Copyright © 2011 Elsevier Inc. All rights reserved.
Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides containing a synthetic abasic lesion (tetrahydrofuran analogue) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, respectively) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.
Copyright © 2010 Elsevier Ltd. All rights reserved.
The predominant organizational theme by which the transcription machinery and chromatin regulators are positioned within promoter regions or throughout genes in a genome is largely unknown. We mapped the genomic location of diverse representative components of the gene regulatory machinery in Saccharomyces cerevisiae to an experimental resolution of <40 bp. Sequence-specific gene regulators, chromatin regulators, mediator, and RNA polymerase (Pol) II were found primarily near the downstream border from the "-1" nucleosome, which abuts against the approximately 140-bp nucleosome-free promoter region (NFR). General transcription factors TFIIA, -B, -D, -E, -F, -H were located near the downstream edge from the NFR. The -1 nucleosome dissociated upon Pol II recruitment, but not upon recruitment of only TBP and TFIIB. The position of many sequence-specific regulators in promoter regions correlated with the position of specific remodeling complexes, potentially reflecting functional interactions. Taken together the findings suggest that the combined action of activators and chromatin remodeling complexes remove the -1 nucleosome after the preinitiation complex (PIC) has partially assembled, but before or concomitant with Pol II recruitment. We find PIC assembly, which includes Pol II recruitment, to be a significant rate-limiting step during transcription, but that additional gene-specific rate-limiting steps associated with Pol II occur after recruitment.
DNA polymerase epsilon (Polepsilon), one of the three major eukaryotic replicative polymerases, is comprised of the essential catalytic subunit, called Pol2 in budding yeast, and three accessory subunits, only one of which, Dpb2, is essential. Polepsilon is recruited to replication origins during late G(1) phase prior to activation of replication. In this work we show that the budding yeast Dpb2 is phosphorylated in a cell cycle-dependent manner during late G(1) phase. Phosphorylation results in the appearance of a lower mobility species. The appearance of that species in vivo is dependent upon the Cdc28 cyclin-dependent protein kinase (CDK), which can directly phosphorylate Dpb2 in vitro. Either G(1) cyclin (Cln) or B-type cyclin (Clb)-associated CDK is sufficient for phosphorylation. Mapping of phosphorylation sites by mass spectrometry using a novel gel-based proteolysis protocol shows that, of the three consensus CDK phosphorylation sites, at least two, Ser-144 and Ser-616, are phosphorylated in vivo. The Cdc28 CDK phosphorylates only Ser-144 in vitro. Using site-directed mutagenesis, we show that Ser-144 is sufficient for the formation of the lower mobility form of Dpb2 in vivo. In contrast, Ser-616 appears not to be phosphorylated by Cdc28. Finally, inactivation of all three CDK consensus sites in Dpb2 results in a synthetic phenotype with the pol2-11 mutation, leading to decreased spore viability, slow growth, and increased thermosensitivity. We suggest that phosphorylation of Dpb2 during late G(1) phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Polepsilon
Recruitment of TATA-binding protein (TBP) is central to activation of transcription by RNA polymerase II (pol II). This depends upon co-activator proteins including TBP-associated factors (TAFs). Yeast Mot1p was identified as a general transcriptional repressor in genetic screens and is also found associated with TBP. To obtain insight into Mot1p function in vivo, we determined the mRNA expression profile of the mot1-1 temperature-sensitive (Ts) strain. Unexpectedly, this indicated that Mot1p mostly plays a positive role for transcription. For one potential activation target, HXT2, we analyzed promoter recruitment of Mot1p, TBP, Taf1p (Taf130p) and pol II by chromatin immunoprecipitation assays. Whereas TBP becomes stably associated upon activation of the HXT2 and HXT4 promoters, Mot1p showed only a transient association. TBP recruitment was compromised in two different mot1 mutant strains, but was only moderately affected in a taf1 Ts strain. Together, our data indicate that Mot1p can assist in recruitment of TBP on promoters during gene activation in vivo.
Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol delta preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K(m)). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol delta preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol delta replication fidelity at 8-oxoG depends upon contributions from K(m), K(d)(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.