Improved detection of DNA replication fork-associated proteins.

Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.

SGS1-SuOff rescues the mild methylmethane sulfonate sensitivity of srs2Δ cells in Saccharomyces cerevisiae.

Sgs1p in Saccharomyces cerevisiae belongs to the RecQ helicase family. Sgs1p is involved in recombination during DNA damage repair and sumoylation of Sgs1p is one mechanism by which the protein is regulated. To further understand the significance of Sgs1p sumoylation in DNA damage repair, we examined the genetic interaction between SGS1 SUMO mutants and a mutant of SRS2, the protein product of which also prevents aberrant recombination structures. We observed that SGS1-SuOff, a mutant in which Sgs1p cannot be sumoylated, attenuates the mild sensitivity of srs2Δcells to methyl methane sulfonate.

Students' Perceptions of FSBio 201, A CURE-Based Course that Scaffolds Research and Scientific Communication, Align with Learning Outcomes.

Incorporating active research opportunities into undergraduate curricula is one of the most cited elements demonstrated to improve inclusive excellence and retention in all STEM fields. Allegheny College has a long and nationally-recognized tradition of collaborative student-faculty research within the academic curriculum and as co-curricular opportunities. We present an example of the former, a Course-based Undergraduate Research Experience (CURE), FSBio 201, that has been central to Allegheny's biology curriculum for over two decades. The course emphasizes biological research design, execution, and communication. We have coded and analyzed feedback from student evaluations and from the national CURE project database, both of which measure students' perceptions and attitudes toward the course. The majority of the student feedback related to the course learning outcomes of fostering independent research and communication skills was positive. However, we also see areas for improvement, such as how we employ peer-to-peer mentoring and how we teach quantitative and computer-based skills. We conclude that students' self-reported data are in line with our learning outcomes and provide FSBio 201 as a model for introducing college undergraduates to biological research.

How Not To Be in the Wrong Place at the Wrong Time: An Education Primer for Use with "Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation".

Recent work by Kentaro Ohkuni and colleagues exemplifies how a series of molecular mechanisms contribute to a cellular outcome-equal distribution of chromosomes. Failure to maintain structural and numerical integrity of chromosomes is one contributing factor in genetic diseases such as cancer. Specifically, the authors investigated molecular events surrounding centromeric histone H3 variant Cse4 deposition-a process important for chromosome segregation, using Saccharomyces cerevisiae as a model organism. This study illustrates an example of a post-translational modification-sumoylation-regulating a cellular process and the concept of genetic interactions (e.g, synthetic dosage lethality). Furthermore, the study highlights the importance of using diverse experimental approaches in answering a few key research questions. The authors used molecular biology techniques (e.g., qPCR), biochemical experiments (e.g., Ni-NTA/8His protein purification), as well as genetic approaches to understand the regulation of Cse4 At a big-picture level, the study reveals how genetic changes can lead to subsequent molecular and cellular changes.

Flap endonuclease overexpression drives genome instability and DNA damage hypersensitivity in a PCNA-dependent manner.

Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.

Slx5/Slx8 Promotes Replication Stress Tolerance by Facilitating Mitotic Progression.

Loss of minichromosome maintenance protein 10 (Mcm10) causes replication stress. We uncovered that S. cerevisiae mcm10-1 mutants rely on the E3 SUMO ligase Mms21 and the SUMO-targeted ubiquitin ligase complex Slx5/8 for survival. Using quantitative mass spectrometry, we identified changes in the SUMO proteome of mcm10-1 mutants and revealed candidates regulated by Slx5/8. Such candidates included subunits of the chromosome passenger complex (CPC), Bir1 and Sli15, known to facilitate spindle assembly checkpoint (SAC) activation. We show here that Slx5 counteracts SAC activation in mcm10-1 mutants under conditions of moderate replication stress. This coincides with the proteasomal degradation of sumoylated Bir1. Importantly, Slx5-dependent mitotic relief was triggered not only by Mcm10 deficiency but also by treatment with low doses of the alkylating drug methyl methanesulfonate. Based on these findings, we propose a model in which Slx5/8 allows for passage through mitosis when replication stress is tolerable.

MCM10: one tool for all-Integrity, maintenance and damage control.

Minichromsome maintenance protein 10 (Mcm10) is an essential replication factor that is required for the activation of the Cdc45:Mcm2-7:GINS helicase. Mcm10's ability to bind both ds and ssDNA appears vital for this function. In addition, Mcm10 interacts with multiple players at the replication fork, including DNA polymerase-α and proliferating cell nuclear antigen with which it cooperates during DNA elongation. Mcm10 lacks enzymatic function, but instead provides the replication apparatus with an oligomeric scaffold that likely acts in the coordination of DNA unwinding and DNA synthesis. Not surprisingly, loss of Mcm10 engages checkpoint, DNA repair and SUMO-dependent rescue pathways that collectively counteract replication stress and chromosome breakage. Here, we review Mcm10's structure and function and explain how it contributes to the maintenance of genome integrity.

Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression.

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.

Enigmatic roles of Mcm10 in DNA replication.

Minichromosome maintenance protein 10 (Mcm10) is required for DNA replication in all eukaryotes. Although the exact contribution of Mcm10 to genome replication remains heavily debated, early reports suggested that it promotes DNA unwinding and origin firing. These ideas have been solidified by recent studies that propose a role for Mcm10 in helicase activation. Whereas the molecular underpinnings of this activation step have yet to be revealed, structural data on Mcm10 provide further insight into a possible mechanism of action. The essential role in DNA replication initiation is not mutually exclusive with additional functions that Mcm10 may have as part of the elongation machinery. Here, we review the recent findings regarding the role of Mcm10 in DNA replication and discuss existing controversies.

Targeting aurora kinases limits tumour growth through DNA damage-mediated senescence and blockade of NF-κB impairs this drug-induced senescence.

Oncogene-induced senescence can provide a protective mechanism against tumour progression. However, production of cytokines and growth factors by senescent cells may contribute to tumour development. Thus, it is unclear whether induction of senescence represents a viable therapeutic approach. Here, using a mouse model with orthotopic implantation of metastatic melanoma tumours taken from 19 patients, we observed that targeting aurora kinases with MLN8054/MLN8237 impaired mitosis, induced senescence and markedly blocked proliferation in patient tumour implants. Importantly, when a subset of tumour-bearing mice were monitored for tumour progression after pausing MLN8054 treatment, 50% of the tumours did not progress over a 12-month period. Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP). Blockade of IKKß/NF-κB led to reversal of MLN8237-induced senescence and SASP. Results demonstrate that removal of senescent tumour cells by infiltrating myeloid cells is crucial for inhibition of tumour re-growth. Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

NF-κB inducing kinase: a key regulator in the immune system and in cancer.

NF-κB inducing kinase (NIK) is a kinase that activates the canonical and non-canonical NF-κB pathways to control transcriptional expression of certain proteins such as cytokines, chemokines and NF-κB signaling molecules. Many advances have been made in understanding the molecular mechanisms by which the stability of NIK is regulated to affect downstream signaling. Genetic mouse models suggest that NIK plays an essential role in the regulation of the immune system as well as in the bone microenvironment. Increasing evidence links NIK to the tumorigenesis of hematological cancers, such as multiple myeloma, and solid tumors, such as pancreatic carcinoma and melanoma. Understanding the mechanism by which NIK is de-regulated will potentially provide therapeutic options for certain diseases such as autoimmunity and cancer.

Molecular determinants of melanoma malignancy: selecting targets for improved efficacy of chemotherapy.

The BRAFV600E mutation is common in human melanoma. This mutation enhances IkappaB kinase (IKK)/nuclear factor-kappaB (NF-kappaB) and extracellular signal-regulated kinase/activator protein signaling cascades. In this study, we evaluated the efficacy of targeting either B-Raf or IKKbeta in combination with the DNA alkylating agent temozolomide for treatment of advanced metastatic melanoma. Xenografts of Hs294T human metastatic melanoma cells exhibiting the BRAFV600E mutation were treated with inhibitors of IKKbeta (BMS-345541), B-Raf (BAY 54-9085), and/or temozolomide. Drug response was mechanistically analyzed in vitro and in vivo. In this study, we determined that the antitumor activity of all three drugs depends on inhibition of NF-kappaB. BMS-345541 inhibits IKKbeta-mediated phosphorylation of IkappaBalpha and thus blocks the nuclear localization of NF-kappaB, whereas BAY 54-9085 inhibits activation of NF-kappaB through a mechanism that does not involve stabilization of IkappaBalpha. Moreover, BMS-345541, but not BAY 54-9085, activates the death pathways of p53 and c-Jun-NH2-kinase, contributing to the killing of melanoma cells. Temozolomide inhibits both NF-kappaB and extracellular signal-regulated kinase activity, conferring effective in vivo antitumor activity. Thus, temozolomide, but not BAY 54-9085, has a synergistic in vivo antitumor effect with BMS-345541. We conclude that the efficacy of antimelanoma therapy depends on inhibition of expression of antiapoptotic genes transcriptionally regulated by NF-kappaB. In contrast, drug targeting of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway alone in melanoma cells is ineffective for melanoma therapy in cases where NF-kappaB is not also targeted.

The lymphotoxin-beta receptor is an upstream activator of NF-kappaB-mediated transcription in melanoma cells.

The pleiotropic transcription factor nuclear factor-kappaB (NF-kappaB (p50/p65)) regulates the transcription of genes involved in the modulation of cell proliferation, apoptosis, and oncogenesis. Furthermore, a host of solid and hematopoietic tumor types exhibit constitutive activation of NF-kappaB (Basseres, D. S., and Baldwin, A. S. (2006) 25, 6817-6830). However, the mechanism for this constitutive activation of NF-kappaB has not been elucidated in the tumors. We have previously shown that NF-kappaB-inducing kinase (NIK) protein and its association with Inhibitor of kappaB kinase alphabeta are elevated in melanoma cells compared with their normal counterpart, leading to constitutive activation of NF-kappaB. Moreover, expression of dominant negative NIK blocked this base-line NF-kappaB activity in melanoma cells. Of the three receptors that require NIK for activation of NF-kappaB, only the lymphotoxin-beta receptor (LTbeta-R) is expressed in melanoma. We show in this manuscript that for melanoma there is a strong relationship between expression of the LTbeta-R and constitutive NF-kappaB transcriptional activity. Moreover, we show that activation of the LTbeta-R can drive NF-kappaB activity to regulate gene expression that leads to enhanced cell growth. The inhibition by LTbeta-R shRNA resulted in decreased NF-kappaB promoter activity, decreased growth, and decreased invasiveness as compared with control. These results indicate that the LTbeta-R constitutively induces NF-kappaB activation, and this event may be associated with autonomous growth of melanoma cells.

Role of chemokines in tumor growth.

Chemokines play a paramount role in the tumor progression. Chronic inflammation promotes tumor formation. Both tumor cells and stromal cells elaborate chemokines and cytokines. These act either by autocrine or paracrine mechanisms to sustain tumor cell growth, induce angiogenesis and facilitate evasion of immune surveillance through immunoediting. The chemokine receptor CXCR2 and its ligands promote tumor angiogenesis and leukocyte infiltration into the tumor microenvironment. In harsh acidic and hypoxic microenvironmental conditions tumor cells up-regulate their expression of CXCR4, which equips them to migrate up a gradient of CXCL12 elaborated by carcinoma-associated fibroblasts (CAFs) to a normoxic microenvironment. The CXCL12-CXCR4 axis facilitates metastasis to distant organs and the CCL21-CCR7 chemokine ligand-receptor pair favors metastasis to lymph nodes. These two chemokine ligand-receptor systems are common key mediators of tumor cell metastasis for several malignancies and as such provide key targets for chemotherapy. In this paper, the role of specific chemokines/chemokine receptor interactions in tumor progression, growth and metastasis and the role of chemokine/chemokine receptor interactions in the stromal compartment as related to angiogenesis, metastasis, and immune response to the tumor are reviewed.