Novel function and regulation of mutagenic DNA polymerases in Escherichia coli

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007.Vita.Includes bibliographical references.The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV-light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel, low to moderate fidelity DNA polymerases with the ability to catalyze synthesis on imperfect DNA templates in a process termed translesion synthesis (TLS). Similar enzymes exist in nearly all organisms, constituting the Y-superfamily of DNA polymerases. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, its precise function has remained elusive. Here we show that AdinB E. coli strains are sensitive to DNA damaging agents that form lesions at the N2 position of guanine. In vitro bypass studies of an N2-guanine adduct by DinB demonstrate considerable preference for correct nucleotide insertion and an increased catalytic proficiency on the lesion-bearing template relative to undamaged DNA. Moreover, DinB and its mammalian and archaeal orthologs possess similar substrate specificities. Mutation of a single residue in the active site ofE. coli DinB suggests that its enhanced activity is coupled to lesion recognition and that its TLS function is required for resistance to DNA damaging agents in vivo.(cont.) Regulation of the mutagenic potential of DinB is critical for maintenance of genomic integrity. We present evidence indicating that abortive TLS products generated by a DinB variant are subject to the proofreading function of DNA polymerase III. Moreover, both the TLS activity and -1 frameshift mutator potential of DinB are modulated in a highly sophisticated manner by the DNA damage-inducible proteins RecA and UmuD2. These biochemical data, coupled with genetic analyses and molecular modeling, indicate that DinB is a specialized and remarkably controlled translesion DNA polymerase. In addition, we present evidence that the umuC+participates in several novel biological functions in addition to its established role in TLS. A novel umuC gain-of-function allele confers striking resistance to hydroxyurea and umuC+ mediates the expression of genes and physiological responses under conditions of SOS induction. Taken together, these observations hint at at a largely uncharacterized function of Y-family polymerases in sculpting physiological responses, including active mechanisms of cell death, in response to environmental stress.by Daniel F. Jarosz.Ph.D

Colloidal topological insulators

Topological insulators insulate in the bulk but exhibit robust conducting edge states protected by the topology of the bulk material. Here, we design a colloidal topological insulator and demonstrate experimentally the occurrence of edge states in a classical particle system. Magnetic colloidal particles travel along the edge of two distinct magnetic lattices. We drive the colloids with a uniform external magnetic field that performs a topologically non-trivial modulation loop. The loop induces closed orbits in the bulk of the magnetic lattices. At the edge, where both lattices merge, the colloids perform skipping orbits trajectories and hence edge-transport. We also observe paramagnetic and diamagnetic colloids moving in opposite directions along the edge between two inverted patterns; the analogue of a quantum spin Hall effect in topological insulators. We present a new, robust, and versatile way of transporting colloidal particles, enabling new pathways towards lab on a chip applications

Cryptic Variation in Morphological Evolution: HSP90 as a Capacitor for Loss of Eyes in Cavefish

In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting

Neurodegenerative Disease and the NLRP3 Inflammasome.

The prevalence of neurodegenerative disease has increased significantly in recent years, and with a rapidly aging global population, this trend is expected to continue. These diseases are characterised by a progressive neuronal loss in the brain or peripheral nervous system, and generally involve protein aggregation, as well as metabolic abnormalities and immune dysregulation. Although the vast majority of neurodegeneration is idiopathic, there are many known genetic and environmental triggers. In the past decade, research exploring low-grade systemic inflammation and its impact on the development and progression of neurodegenerative disease has increased. A particular research focus has been whether systemic inflammation arises only as a secondary effect of disease or is also a cause of pathology. The inflammasomes, and more specifically the NLRP3 inflammasome, a crucial component of the innate immune system, is usually activated in response to infection or tissue damage. Dysregulation of the NLRP3 inflammasome has been implicated in the progression of several neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases. This review aims to summarise current literature on the role of the NLRP3 inflammasome in the pathogenesis of neurodegenerative diseases, and recent work investigating NLRP3 inflammasome inhibition as a potential future therapy

Biomolecular Condensation: A New Phase in Cancer Research

UNLABELLED: Multicellularity was a watershed development in evolution. However, it also meant that individual cells could escape regulatory mechanisms that restrict proliferation at a severe cost to the organism: cancer. From the standpoint of cellular organization, evolutionary complexity scales to organize different molecules within the intracellular milieu. The recent realization that many biomolecules can phase-separate into membraneless organelles, reorganizing cellular biochemistry in space and time, has led to an explosion of research activity in this area. In this review, we explore mechanistic connections between phase separation and cancer-associated processes and emerging examples of how these become deranged in malignancy. SIGNIFICANCE: One of the fundamental functions of phase separation is to rapidly and dynamically respond to environmental perturbations. Importantly, these changes often lead to alterations in cancer-relevant pathways and processes. This review covers recent advances in the field, including emerging principles and mechanisms of phase separation in cancer

Soft-core processor study for node-based architectures.

Node-based architecture (NBA) designs for future satellite projects hold the promise of decreasing system development time and costs, size, weight, and power and positioning the laboratory to address other emerging mission opportunities quickly. Reconfigurable Field Programmable Gate Array (FPGA) based modules will comprise the core of several of the NBA nodes. Microprocessing capabilities will be necessary with varying degrees of mission-specific performance requirements on these nodes. To enable the flexibility of these reconfigurable nodes, it is advantageous to incorporate the microprocessor into the FPGA itself, either as a hardcore processor built into the FPGA or as a soft-core processor built out of FPGA elements. This document describes the evaluation of three reconfigurable FPGA based processors for use in future NBA systems--two soft cores (MicroBlaze and non-fault-tolerant LEON) and one hard core (PowerPC 405). Two standard performance benchmark applications were developed for each processor. The first, Dhrystone, is a fixed-point operation metric. The second, Whetstone, is a floating-point operation metric. Several trials were run at varying code locations, loop counts, processor speeds, and cache configurations. FPGA resource utilization was recorded for each configuration. Cache configurations impacted the results greatly; for optimal processor efficiency it is necessary to enable caches on the processors. Processor caches carry a penalty; cache error mitigation is necessary when operating in a radiation environment