Biochemical characterization of FANCD2 and FANCM in the Fanconi Anaemia pathway

Abstract : The maintenance of genome stability is a challenge for all living organisms. DNA is regularly subjected to chemical alterations by both endogenous and exogenous DNA damaging agents. If left unrepaired, these lesions will create mutations or lead to chromosomal instability. DNA crosslinking agents probably bring about the most toxic lesions. By linking covalently the two strands of DNA, crosslinking agents will impede essential cellular processes such as replication and transcription. Cells from Fanconi anaemia patients are extremely sensitive to these agents. Fanconi anaemia (FA) is a rare chromosomal instability disorder that leads to developmental defects, pancytopenia and cancer susceptibility. FA is a genetically heterogeneous disease with thirteen complementation groups identified. Proteins encoded by the FA genes work together in the FA pathway. Eight of these proteins form the FA core complex (FANC-A, B, C,E, F, G, L and -M), whose integrity is required to monoubiquitinate FANCD2 and FANCI in response to DNA damage. The hypersensitivity of FA cells to crosslinking agents, which perturb the progression of replication forks, has led to the hypothesis that FA proteins play a crucial role in the response to replication stress. However, at the molecular level, the functions of the FA pathway remain largely unknown. Our efforts were first focused on the characterization of FANCD2, "the key effector of the FA pathway". Using different substrates, we found that in vitro, purified hFANCD2 preferentially binds single strand DNA and double strand DNA extremities. Concomitantly, FANCM was identified as a new component of the FA core complex. Moreover FANCM was shown to have specific branch migration activities and probably a role as a "landing platform" on DNA for the other components of the core complex. By using FANCM mutants carrying deletions within the internal domain, we investigated the role of FANCM as a DNA anchor protein for the core complex. We observed that indeed, a specific part of the internal domain of FANCM interacts with components of the core complex. Finally, in collaboration with Weidong Wang's lab we characterized two new components of the FA pathway: FAAP10 and FAAP16. As a heterodimer these two proteins show affinity for dsDNA, and anneal complementary oligonucleotides in vitro. Moreover these proteins can associate with FANCM via a part of its internal domain. We find that FANCM, FAAP 10 and FAAP 16 can co-exist on the branch point of replication and recombination intermediates, and that FAAP10 and FAAP16 stimulate replication fork reversal by FANCM. These results suggest that FANCM may function as a landing platform for the core complex. After loading on DNA, the core complex can activate FANCD2 through monoubiquitination leading to its recruitment to the site of damage. Since ssDNA and double strand breaks are intermediates that are generated as a consequence of collapsed replication forks, FANCD2 by binding to ds DNA ends and ssDNA could protect such structures from the recombination repair machinery and prevent unscheduled recombination events. Alternatively, FANCD2 could avoid nucleases from gaining access to collapsed forks, preserving the DNA in state that can be used as a starting point for resumption of DNA synthesis. The overall comprehension of the FA pathway is far from been complete. Our results unravel new aspects of Fanconi Anaemia, which hopefully in the near future will address keys questions leading to a better understanding of the fascinating Fanconi Anaemia. Résumé : Le maintien de l'intégrité du génome est fondamentale chez tous les organismes vivants. L'ADN est constamment altéré par des composés aussi bien endogènes qu'exogènes. Si ces altérations ne sont pas réparées, elles peuvent conduire à l'apparition de mutations, ainsi qu'à une instabilité génomique accrue. Les lésions les plus sévères qui peuvent survenir sur l'ADN, sont les pontages inter caténaires. Des agents pontants en liant de façon covalente les deux brins d'ADN, vont empêcher le déroulement normal de processus cellulaires essentiels tels que la réplication ou la transcription. La compréhension des mécanismes permettant à la cellule de tolérer et réparer ces lésions est primordiale, notamment dans le cas des patients atteints de l'anémie de Fanconi qui présentent une très grande sensibilité à ces composés pontants. L'anémie de Fanconi est une maladie génétique rare appartenant à un groupe de pathologies associées à une grande instabilité chromosomique. Les patients atteints de l'anémie de Fanconi présentent des malformations du squelette, une pancytopénie et une forte propension à la survenue de cancer. L'anémie de Fanconi est génétiquement très hétérogène. À ce jour, 13 gènes codant pour 13 protéines FANC différentes ont été identifiés. Huit de ces protéines fonctionnent ensemble au sein d'un complexe (nommé le complexe FANC) ayant pour but de monoubiquitiner FANCD2 et FANCI en réponse à la formation de lésions sur l'ADN. L'extrême sensibilité des cellules de patients atteints de l'anémie de Fanconi à ces agents pontant l'ADN suggère l'implication des protéines FANC dans la réponse cellulaire suite à une stress réplicatif. Cependant, le rôle moléculaire exact de ces protéines demeure encore inconnu. Après purification, nous avons observé que FANCD2 était capable de lier l'ADN simple brin, ainsi que les extrémités d'ADN in vitro. Dans le même temps, FANCM fut identifié comme appartenant au complexe FANC. FANCM est décrit comme une translocase capable de promouvoir le déplacement de point de jonction dans des structures d'ADN spécifiques in vitro. De plus, en se liant à l'ADN, FANCM peut agir comme une plateforme pour les autres protéines FANC, leur permettant ainsi d'être adressées à l'ADN. En créant des protéines FANCM recombinantes ayant des délétions dans le domaine interne, nous avons pu observer que certaines protéines du complexe FANC se fixent à des sites spécifiques sur le domaine interne de FANCM. Enfin, au travers d'une collaboration, nous avons été amenés à caractériser deux nouvelles protéines appartenant au complexe FANC : FAAP 10 et FAAP16. Elles s'associent à FANCM par l'intermédiaire du domaine interne, et forment ainsi un hétérotrimére. La présence de FAAP10 et FAAP16 n'affecte pas la liaison de FANCM à l'ADN, mais semble potentialiser son activité de régression in vitro. FANCM semble donc fonctionner comme une plateforme pour les autres composants du complexe FANC. Ces derniers, une fois liés à l'ADN permettent la monoubiquitination de FANCD2 et son recrutement au site lésé de l'ADN. FANCD2 en se liant de façon préférentielle à l'ADN simple brin et aux extrémités d'ADN qui sont générés lors de l'arrêt et du démantèlement d'une fourche de réplication, pourrait protéger ces même fourches de réplication arrêtées, d'évènements de recombinaison aléatoires. Nos résultats apportent de nouveaux éléments concernant les mécanismes moléculaires de l'anémie de Fanconi. Enfin, l'étude de l'anémie de Fanconi permet aussi de mieux comprendre les mécanismes mis en place par la cellule pour tolérer des lésions survenant lors de la réplication.

Comparison of microsatellite instability and chromosomal instability in predicting survival of patients with T3N0 colorectal cancer.

BACKGROUND: At least 2 apparently independent mechanisms, microsatellite instability (MSI) and chromosomal instability, are implicated in colorectal tumorigenesis. Their respective roles in predicting clinical outcomes of patients with T3N0 colorectal cancer remain unknown. METHODS: Eighty-eight patients with a sporadic T3N0 colon or rectal adenocarcinoma were followed up for a median of 67 months. For chromosomal instability analysis, Ki-ras mutations were determined by single-strand polymerase chain reaction, and p53 protein staining was studied by immunohistochemistry. For MSI analysis, DNA was amplified by polymerase chain reaction at 7 microsatellite targets (BAT25, BAT26, D17S250, D2S123, D5S346, transforming growth factor receptor II, and BAX). RESULTS: Overall 5-year survival rate was 72%. p53 protein nuclear staining was detected in 39 patients (44%), and MSI was detected in 21 patients (24%). MSI correlated with proximal location (P <.001) and mucinous content (P <.001). In a multivariate analysis, p53 protein expression carried a significant risk of death (relative risk = 4.0, 95% CI = 1.6 to 10.1, P =.004). By comparison, MSI was not a statistically significant prognostic factor for survival in this group (relative risk = 2.2, 95% CI = 0.6 to 7.3, P =.21). CONCLUSIONS: p53 protein overexpression provides better prognostic discrimination than MSI in predicting survival of patients with T3N0 colorectal cancer. Although MSI is associated with specific clinicopathologic parameters, it did not predict overall survival in this group. Assessment of p53 protein expression by immunocytochemistry provides a simple means to identify a subset of T3N0 patients with a 4-times increased risk for death.

Feasibility of Visualization and Simulation Applications to Improve Work Zone Safety and Mobility

Visualization is a relatively recent tool available to engineers for enhancing transportation project design through improved communication, decision making, and stakeholder feedback. Current visualization techniques include image composites, video composites, 2D drawings, drive-through or fly-through animations, 3D rendering models, virtual reality, and 4D CAD. These methods are used mainly to communicate within the design and construction team and between the team and external stakeholders. Use of visualization improves understanding of design intent and project concepts and facilitates effective decision making. However, visualization tools are typically used for presentation only in large-scale urban projects. Visualization is not widely accepted due to a lack of demonstrated engineering benefits for typical agency projects, such as small- and medium-sized projects, rural projects, and projects where external stakeholder communication is not a major issue. Furthermore, there is a perceived high cost of investment of both financial and human capital in adopting visualization tools. The most advanced visualization technique of virtual reality has only been used in academic research settings, and 4D CAD has been used on a very limited basis for highly complicated specialty projects. However, there are a number of less intensive visualization methods available which may provide some benefit to many agency projects. In this paper, we present the results of a feasibility study examining the use of visualization and simulation applications for improving highway planning, design, construction, and safety and mobility.

Children's Mental Health and Well-Being Work-group Final Report, December 15, 2015

The children's mental health and well-being work group was formed in response to legislative direction to facilitate a study and make recommendations regarding children's mental health and the systems that assist children and families in Iowa.

Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

Work Zone Simulation Model, 1999

To support the analysis of driver behavior at rural freeway work zone lane closure merge points, Center for Transportation Research and Education staff collected traffic data at merge areas using video image processing technology. The collection of data and the calculation of the capacity of lane closures are reported in a companion report, "Traffic Management Strategies for Merge Areas in Rural Interstate Work Zones". These data are used in the work reported in this document and are used to calibrate a microscopic simulation model of a typical, Iowa rural freeway lane closure. The model developed is a high fidelity computer simulation with an animation interface. It simulates traffic operations at a work zone lane closure. This model enables traffic engineers to visually demonstrate the forecasted delay that is likely to result when freeway reconstruction makes it necessary to close freeway lanes. Further, the model is also sensitive to variations in driver behavior and is used to test the impact of slow moving vehicles and other driver behaviors. This report consists of two parts. The first part describes the development of the work zone simulation model. The simulation analysis is calibrated and verified through data collected at a work zone in Interstate Highway 80 in Scott County, Iowa. The second part is a user's manual for the simulation model, which is provided to assist users with its set up and operation. No prior computer programming skills are required to use the simulation model.

Traffic Control in Work Zones with Edge Drop-Offs, 2002

Pavement and shoulder edge drop-offs commonly occur in work zones as the result of overlays, pavement replacement, or shoulder construction. The depth of these elevation differentials can vary from approximately one inch when a flexible pavement overlay is applied to several feet where major reconstruction is undertaken. The potential hazards associated with pavement edge differentials depend on several factors including depth of the drop-off, shape of the pavement edge, distance from traveled way, vehicle speed, traffic mix, volume, and other factors. This research was undertaken to review current practices in other states for temporary traffic control strategies addressing lane edge differentials and to analyze crash data and resultant litigation related to edge drop-offs. An objective was to identify cost-effective practices that would minimize the potential for and impacts of edge drop crashes in work zones. Considerable variation in addressing temporary traffic control in work zones with edge drop-off exposure was found among the states surveyed. Crashes related to pavement edge drop-offs in work zones do not commonly occur in the state of Iowa, but some have resulted in significant tort claims and settlements. The use of benefit/cost analysis may provide guidance in selection of an appropriate mitigation and protection of edge drop-off conditions. Development and adoption of guidelines for design of appropriate traffic control for work zones that include edge drop-off exposure, particularly identifying effective use of temporary barrier rail, may be beneficial in Iowa.

Maintenance of Traffic for Innovative Geometric Design Work Zones, 2015

Currently there are no guidelines within the Manual on Uniform Traffic Control Devices (MUTCD) on construction phasing and maintenance of traffic (MOT) for retrofit construction and maintenance projects involving innovative geometric designs. The research presented in this report addressed this gap in existing knowledge by investigating the state of the practice of construction phasing and MOT for several types of innovative geometric designs including the roundabout, single point urban interchange (SPUI), diverging diamond interchange (DDI), restricted-crossing left turn (RCUT), median U-turn (MUT), and displaced left turn (DLT). This report provides guidelines for transportation practitioners in developing construction phasing and MOT plans for innovative geometric designs. This report includes MOT Phasing Diagrams to assist in the development of MOT strategies for innovative designs. The MOT Phasing Diagrams were developed through a review of literature, survey, interviews with practitioners, and review of plans from innovative geometric design projects. These diagrams are provided as a tool to assist in improving work zone safety and mobility through construction of projects with innovative geometric designs. The aforementioned synthesis of existing knowledge documented existing practices for these types of designs.

Traffic Management Strategies for Merge Area in Rural Interstate Work Zones, CTRE 97-12, 1999

The Iowa Department of Transportation, like many other state transportation agencies, is experiencing growing congestion and traffic delays in work zones on rural interstate highways. The congestion results in unproductive and wasteful delays for both motorists and commercial vehicles. It also results in hazardous conditions where vehicle stopped in queues on rural interstate highways are being approached by vehicles upstream at very high speeds. The delays also result in driver frustration, making some drivers willing to take unsafe risks in an effort to bypass delays. To reduce the safety hazards and unproductive delays of congested rural interstate work zones, the Iowa Department of Transportation would like to improve its traffic management strategies at these locations. Applying better management practices requires knowledge of the traffic flow properties and driver behavior in and around work zones, and knowledge of possible management strategies. The project reported here and in a companion report documents research which seeks to better understand traffic flow behavior at rural interstate highway work zones and to estimate the traffic carrying capacity of work zone lane closures. In addition, this document also reports on technology available to better manage traffic in and around work zones.

Work-Related Exposures to Pesticides in Iowa, 2008-2012

The Pesticide Poisoning Surveillance Program within the Division of ADPER & EH monitors, collects, and analyzes pesticide poisonings to determine the extent to which Iowans are being affected by pesticide exposure. The information gathered by this program is disseminated to governmental agencies, the public, and health care professionals. In addition, IDPH is required to submit its findings annually to the Iowa Department of Agriculture and Land Stewardship (IDALS).

Lessons Learned: Acute Mercury Poisoning From A Residential Work/Hobby Exposure In Iowa, 2014

This document is about the different ways Mercury can poison a person and the exposure in Iowa.