Susan T. Lovett - US grants

DNA binding protein; protein structure function; phosphodiesterase I; Escherichia coli; genetic recombination; bacterial genetics; bacterial proteins; enzyme mechanism; mutant; gene expression; enzyme substrate; immunochemistry;

Cells employ numerous DNA repair and mutation avoidance mechanisms to protect genetic integrity. In the absence of these important processes, cells suffer mutations, chromosomal aberrations or death. Repair-deficient human syndromes have been identified and include neurological and immunological defects, cancer-proneness and premature aging. One common feature of many DNA repair and mutation avoidance mechanisms is the degradation of DNA, accomplished by DNA exonuclease proteins. Exonucleases excise offending DNA lesions or replication errors and promote recombinational repair of broken chromosomes. Exonucleases also prevent inappropriate genetic rearrangements that lead to mutation. Exonucleases produce molecular signals for cell division arrest when the cell is confronted with DNA damaged. A molecular understanding of DNA recombination, repair and mutagenesis will require knowledge of the exonucleases that participate in these processes. Our objective is to define recombination and repair exonucleases of E. coli and Saccharomyces cereviseae. We seek to understand their biochemical properties, their molecular partners and what roles they play in vivo. The RecJ exonuclease from E. coli has been the focus of much of our previous investigation. We have shown that RecJ is a member of a large family of proteins found in archaebacteria, eubacteria and eukaryotes. We will continue to analyze the structure and function of this protein. Physical or functional interactions of RecJ exonuclease with other proteins involved in DNA replication or repair will be assayed. We have identified two new exonucleases from the bacterium E. coli. We will continue to characterize their biochemistry and will analyze mutants in these exonucleases for genetic stability, recombination and DNA repair defects. As it is clear that some of these functions are genetically redundant, multiple mutants in these and other genes will assessed for genetic properties. Physical monitoring of DNA repair and assessment of SOS regulation will be performed in ssExo mutants. A third putative DNA exonuclease from E. coli will be assayed for activity on oligonucleotides. Mutants and genetic suppressors of this function will be characterized. We will investigate the role of putative 3' exonucleases (based on sequence similarity) from the yeast Saccharomyces cerevisiae. The genes will be expressed in E. coli to verify if they encode exonucleases. Mutants in conservied residues will be examined for biological effects.

DESCRIPTION: Sequence analysis of deletion mutations in both E. coli and humans has shown that deletions occur most frequently between short repeated sequences. Models to explain this observation have been of two main types: recombinational (unequal crossing over) and replicational (strand slippage or misalignment). There has been a tendency to discount recombinational mechanisms because deletion mutations in E. coli lack two traditional hallmarks of homologous recombination: the repeated sequences are very often much shorter than necessary to serve as a substrate for RecA protein, and the deletions occur quite readily in RecA deletion strains, in which conventional recombination is eliminated. Recent work by Dr. Lovett using a plasmid-based system for monitoring deletions has shown, unexpectedly, that RecA-independent deletion formation can have unmistakable recombinational features, most notably, deletion-associated plasmid dimerization. On the basis of this and related observations, Dr. Lovett has proposed a mechanism for deletion formation initiated by RecA-independent pairing between nascent strands in the vicinity of an arrested replication fork to form a Holiday junction, followed by processing by other enzymes associated with homologous recombination reactions. Dr. Lovett furthermore proposes that the same mechanism may apply to recombinational "postreplication" repair observed on damaged DNA templates, whose mechanism has been obscure. In this application, Dr. Lovett proposes to continue her study on the mechanism of deletion formation and its relationship to recombinational DNA repair and sister strand exchange (plasmid dimerization) in E. coli. The proposed work has two broad components. In one component, Dr. Lovett will analyze the effect of single DNA lesions (thymine dimers) on the occurrence of deletions and plasmid dimerization.Using variations of this basic idea, she will examine if a thymine dimer has different effects when placed in the leading vs. lagging strand vs. both together; when placed in different locations relative to the tandemly repeated sequences that are recombining; and when processed in strains with mutations in various components of recombination or replication. In the other part of the investigation, Dr. Lovett will define the genetics of recA-independent deletion formation by examining the effects of known components of replication and recombination on deletion formation, and by searching for novel genes that affect the frequency of deletion formation.

DESCRIPTION (provided by applicant): Our specific aims seek to extend our knowledge about the role of nucleases in recombination, repair and mutation avoidance. Maintenance of genomic stability is important to all organisms and defects in these processes lead to cancer-predisposition in several defined human syndromes. Many of the mechanisms that ensure genetic stability are conserved in prokaryotes and eukaryotes. Our studies will employ both biochemical and genetic approaches using the bacterium E. coli. 1. RecJ is a 5' ssDNA exonuclease involved in DNA recombination, repair and mutation avoidance. It is the best-characterized member of a large group of proteins with a novel structure. Our first aim will be to examine some questions raised by the crystal structure of RecJ, namely, the nature of the metal binding site, its interaction with DNA and the structural basis of processivity. 2. Our second aim seeks elucidate the mechanism of template-switch mutations in quasipalindromes and illegitimate recombination reactions at very short homologies. We have shown that both processes are normally circumvented by single-strand DNA exonucleases. Both types of processes may contribute significantly to mutagenesis and genomic evolution. We will develop assays to examine cis-and trans-acting factors on these mutagenic processes. Other experiments to find natural vulnerable sites for such mutations will help establish how these processes contribute to mutational burden of E. coli. 3. Our third aim is to pursue the genetic and biochemical function of the bacterial RadC protein. This protein shares a duplicated helix-hairpin-helix motif characteristic of proteins that bind distorted DNA, particularly the Radl/XPF/Mus81 class of proteins that cleave branched structures in DNA. We will examine RadC's activity on a variety of branched structures and its potential functional and physical interaction with other proteins. We will extend genetic analysis of radC to determine what role it may play in repair and recombination.

[unreadable] DESCRIPTION (provided by applicant): How cell cycle events are controlled with growth remains an important and perhaps the most important, outstanding question in prokaryotic biology. A group of conserved and essential GTPase proteins, related to Ras, have been implicated in cell cycle control but remain poorly characterized. The study of the Obg GTPase has the potential to elucidate aspects of the mechanism of chromosome segregation in bacteria, which is not, at present, understood. Because Obg is universally conserved and essential, its function has impacts on all bacteria, including bacterial pathogens, and constitutes a potential target for antibiotic therapy. All eukaryotic cells possess Obg, too-its function may be essential for mitochondria-and therefore its role in eukaryotic cell biology will be important to understand and will be facilitated by studies first in prokaryotes. An integrative approach, combining cell visualization, genetics, biochemistry and physiology, provides an opportunity to make headway into understanding these important and complex problems. The connection between translational stress and cell cycle will be investigated. The hypothesis that SeqA binding to the chromosome controls access to replication initiation and chromosome segregation machinery will be tested. Additional factors in the stringent response control of cell cycle will be sought. Using state-of- the-art fluorescence microscopy, the localization of the E. coli Obg protein will be examined, as well as its impact on bacterial cytoskeleton, including the actin-related MreB helical filament, and the organization of the chromosome and replisome. The interaction of the ObgE protein with several proteins, including those controlling replication initiation and chromosome segregation, processes on which Obg may exert control, will be probed. Several genetic screens will explore the role of ObgE and the DNA binding protein, SeqA in the regulation of DNA replication and chromosome segregation. Finally, the association of ObgE with the ribosome will be studied, as well as the impact of the stringent response and ObgE on ribosome function and stability. LAY DESCRIPTION: We will study a protein present in all bacteria that appears to control their growth. This will provide important missing information about how bacteria divide and may provide a target for antibiotics. [unreadable] [unreadable] [unreadable]

All cells need to minimize changes to their genetic information. The potential for genetic change is not uniform across the genome: some sequences are intrinsically more mutable. Mutational hotspots are found in imperfect inverted repeat sequences from bacteria to humans. The mechanism for mutagenesis at these sites involves a polymerase template switch, whereby the replicating strand realigns and the mutational changes are templated from other sequences in the inverted repeat. This process may drive the formation of inverted repeat sequences, abundant in many genomes. Despite the prevalence and universality of this process, little is known about factors that promote mutation or act to avoid them. Using a natural mutational hotspot in the thyA gene of Escherichia coli, this project will identify the factors that control template-switch mutagenesis, a process that likely impacts all genomes. The unusually high rate of mutagenesis at this site provides a unique opportunity to screen for genes that influence hotspot mutagenesis. This approach will provide new insights into the mechanism of mutagenesis and mutation avoidance. Other experiments seek to clarify whether chromosomal context or damage to the replication fork affects the frequency of such events. Undergraduate and graduate students will perform the proposed experiments and the laboratory has a strong track record of the participation of undergraduate, as well as graduate, students in research. A broader impact of the proposal, therefore, is the training of students in state-of-the art genetic analysis and their mentorship for careers in research and education in the public or private sector.

DESCRIPTION (provided by applicant): Recombinational repair is important for cell survival and stability of genetic material. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. Recent evidence suggests that, in all cells, every round of replication requires some form of replication fork repair. In this proposed study, we will address several important questions in prokaryotic and eukaryotic recombination that have relevance to replication fork repair in every organism. (1) How do the RecA paralog proteins facilitate recombination? Every organism appears to employ a RecA-/Rad51 orthologous strand exchange protein and at least one other RecA/Rad51 paralog protein. What are the paralogs doing? We will use a combined biochemical and genetic approach to address the role of the RecA paralog protein, RadA/Sms in genetic recombination of E. coli. (2) What branched DNA molecules are intermediates of recombination and which enzymes resolve them? Our knowledge of the enzymes that process branched DNA intermediates of recombination is incomplete. We will assay whether the RuvC-related protein of E. coli, YqgF, is involved in recombinational repair and can catalyze cleavage of branched molecules predicted by recombination mechanisms. (3) What factors mediate template-switch repair? This is a recombinational mechanism that leads to sister chromosome exchange without the requirement for a strand-exchange protein such as RecA. Our previous work identified the first two factors involved in template-switch repair of E. coli, the chaperone DnaK and the gamma/tau subunit of DNA polymerase III, DnaX. What other factors enable this reaction? Using genetic analysis, we will test the involvement of proteins of the replisome and seek factors presently unknown. Replication fork repair is important for cell survival and stability of genes. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. This proposal seeks to understand the mechanisms of replication fork repair.

? DESCRIPTION (provided by applicant): This project will support the training of eight students annually at Brandeis University in the field of genetics, most of whom will be appointed in the second year of their Ph. D. training. Students will be engaged in genetics research relating to molecular, cell, and developmental biology and neuroscience, with relevance to the mechanisms and treatment of human disease. The Training Grant Faculty are drawn from highly collaborative and interdisciplinary researchers in the Departments of Biology, Biochemistry and Chemistry. Students work in well-funded and productive laboratories that are supported by recently upgraded core facilities in DNA and protein analysis, proteomics, genomics, microscopy and mouse and viral transgenics. The proposed program emphasizes rigorous training to develop research and other skills including scientific literacy, writing and oal communication and quantitative approaches. The Ph. D. program has a core curriculum of molecular biology, cell biology and ethics and advanced genetics courses, which includes molecular genetics, neurogenetics, population genetics and genomics, epigenetics and human genetics. These core courses are supplemented by elective courses in biochemistry, structural biology, developmental biology, mathematical modeling or neuroscience and courses concerning human diseases such as cancer, infectious disease, neurological and development disorders. Trainees are appointed based on the strength of their academic records and research potential and are supported for two years. Their progress is closely monitored by a committee of Training Grant Faculty selected for each student. Qualifying examinations at the end of the first and second years provide a means to evaluate each student's ability to frame questions and propose research solutions in their emerging area of expertise and in an outside field. The training of students is supplemented by seminars and journal clubs, featuring a wide variety of successful investigators in diverse areas of biological research. Professional development activities feature individual development plans and valuable personal discussions that assist each student's career planning. A new facet of the program is the expansion of skills workshops, aimed at enhancing trainees' master of specific quantitative approaches and fellowship/grant writing skills. Special opportunities for Trainees include enhanced personal interactions with speakers and participation in an annual Genetics Symposium. In addition, Trainees work with Training faculty in the planning and implementation of these activities. The program is assessed yearly through online surveys and personal discussions with Trainees. This, and the small size and interconnectedness of students and faculty, provides training responsive to the needs of each student in a first-class research setting.

Biological Sciences (61)

In the laboratory course being developed by this project students combine genetics and genomics techniques to explore genetic variation within populations. It is based on a pilot course introduced in 2007. Students isolate random E. coli transposon mutations affecting rates of genetic variation and analyze the unique mutants they have isolated to discover and understand functions essential to genetic stability. They then integrate their findings with public domain genomic information resources to develop a Web page for each gene investigated. As a finale for the course, students design their own simple experiment regarding mutagenesis and refine the experiment from the results of their preliminary analysis. Students are assessed, before and after the course, for their level of mastery of basic cellular and molecular processes and for their attitudes towards and understanding of scientific research. In addition, students evaluate the value of various aspects of the course, to aid in its future refinements.

The intellectual merit of this project is that it provides real research laboratory experience in a course that leads to understanding of core concepts in genetics.

The broader impact of this project is that the course serves as a model for future development of interdisciplinary project laboratories at Brandeis University and elsewhere. Course materials (information, protocols, genomic resources, exercises, design, strains) are being made available publicly. The novel integration of genomic analysis with readily accessible experiments with bacteria provides a course paradigm that can be replicated in diverse academic settings.

This REU Site award to Brandeis University, located in Waltham, MA will support the training of 10 students for 10 weeks during the summers of 2017- 2019 in biological research employing modern cell and molecular visualization techniques. This program, encompassing over 50 faculty in the Life Sciences, will introduce undergraduate students to a broad range of topics concerning biological structure and function. Students will conduct full-time research guided by their mentors and will participate in weekly lunch seminars, which will include faculty research presentations and professional development activities such as panel discussion with students and postdoctoral fellows from the Greater Boston area concerning careers in biotechnology, research, education, and policy. There will be group discussions of ethical issues and mentoring in science. Students will write a synopsis of their summer project, with feedback and editing, and will participate in a capstone symposium including poster presentations. Participants will be selected from a nation-wide pool based on academic record, recommendations, and potential for research in biology and should be current freshmen, sophomores or juniors at a college or university.

It is anticipated that a total of 30 students, primarily from schools with limited research opportunities, will be trained in the program which welcomes students from underrepresented groups in science. Training will take place in a supportive and interactive environment, and the participating faculty have a strong record of mentoring undergraduates in research and publishing with student co-authors. Students will have an opportunity to interact with scientists with diverse interests, at different stages in their careers, to learn how research is done. Many students will present their work at scientific conferences.

A common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. Students will be tracked after the program in order to determine their career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://www.bio.brandeis.edu/undergrad/summerResearch/ or by contacting the PI (Dr. Susan Lovett at lovett@brandeis.edu) or the co-PI (Dr. Paradis at paradis@brandeis.edu).

This proposal focuses on several mutational changes accompanying DNA repair in budding yeast. DNA repair is induced by galactose-regulated expression of the site-specific HO endonuclease, creating a single double-strand break (DSB). One major goal is to understand complex mutations associated by template switching that occur during gene conversion. Two types of repair will be studied: 1. Quasipalindrome mutation formation during gene conversion and 2. Interchromosomal microhomology-mediated template switching during gene conversion. Genetic analysis of helicases and other repair factors will be screened to find proteins that control the level of these two events, along with an exploration of the role of chromatin. A second goal is to understand changes in repeat copy number during gene conversion, motivated by our recent discovery of important differences between DSB break repair and gap repair. In collaboration with Mitch McVey, another member of this Program Project who focuses on DSB repair in fruit flies, we will assess the frequency of abortive gap repair leading to deletions between repeated sequences within the copied region.

This proposal focuses on several mutational changes accompanying DNA repair in budding yeast. DNA repair is induced by galactose-regulated expression of the site-specific HO endonuclease, creating a single double-strand break (DSB). One major goal is to understand complex mutations associated by template switching that occur during gene conversion. Two types of repair will be studied: 1. Quasipalindrome mutation formation during gene conversion and 2. Interchromosomal microhomology-mediated template switching during gene conversion. Genetic analysis of helicases and other repair factors will be screened to find proteins that control the level of these two events, along with an exploration of the role of chromatin. A second goal is to understand changes in repeat copy number during gene conversion, motivated by our recent discovery of important differences between DSB break repair and gap repair. In collaboration with Mitch McVey, another member of this Program Project who focuses on DSB repair in fruit flies, we will assess the frequency of abortive gap repair leading to deletions between repeated sequences within the copied region.

The overall goal of this project is to study how cells maintain the stability of their genomic DNA. Mutations can arise in genomes due to natural failures during DNA replication or can be induced by environmental factors such as radiation and certain chemicals; mutations promote cancer, aging, neurodegenerative and other diseases, hence it is important to understand their genesis and processing. Some frequent “hotspot” mutations arise during stalled replication, when the nascent DNA strand wanders and finds a wrong strand to copy. This “template-switch” event is the main subject of this project. The aims are to determine what genes influence the frequency of such mutation events and what chemicals or other environmental factors might promote them. A broader impact goal is to train students from diverse backgrounds on how to conduct scientific research, using a simple organism, budding yeast, that can be easily grown and genetically manipulated in the laboratory. A project lab will be developed to introduce students with no prior experience to research practice and techniques, in a supportive and mentored environment.

Genetic assays for template-switch mutations at imperfect inverted repeats in DNA have been engineered in yeast and will be used to identify genes that influence mutagenesis. Preliminary evidence suggests that double-strand DNA breaks frequently undergo template-switching during repair. The HO endonuclease-induced mating-type switching system will be used to study template-switching during DNA replication events that accompany double-strand break repair. Template-switching during normal replication will also be examined for comparison. Finally, potential mutagens will be screened using the template-switch reporter system. The outcomes will increase knowledge about the mechanisms of mutagenesis and its avoidance, and provide valuable information about DNA processing associated with replication difficulties and how replication is coordinated with repair.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The overall goal of this project is to develop tools to study how a common genetic lesion is repaired and removed from the cell. One of the most frequent assaults to genetic material is the formation of DNA-protein crosslinks, covalent bonds that form between DNA and protein. Some of these are unavoidable consequences of cellular metabolism, including the processing of alcohol, and others can be induced by environmental exposure or pharmaceutical agents. These lesions interfere with DNA replication and can cause genetic mutations with deleterious consequences to cell survival. In humans, DNA-protein crosslinks have been implicated in causing cancer and premature aging. Understanding how DNA-protein crosslinks are repaired will allow us to develop ways to avoid the problems associated with these lesions or to enhance their removal. A broader impact of this project is to train students from diverse backgrounds on how to conduct scientific research.

Producing a DNA protein crosslink at a particular genetic site and at a particular time would allow investigators to study the events that that place to achieve crosslink repair. This project will use proteins that naturally produce crosslinks to DNA, deoxycytosine methyl-transferases (CMeTs), which can be trapped after addition of a drug, 5-azacytidine. The study will employ model genetic organisms, brewer’s yeast (Saccharomyces cerevisiae) and a gut bacterium (Escherichia coli), which can be grown easily in the laboratory and for which a variety of genome manipulation tools are available. To achieve site-specificity, CMeT from bacterial restriction systems will be engineered for expression in both model organisms. There are plans to engineer a dCAS9-CMeT as well, in which site-specificity can be conferred by expression of a guide RNA. One CMeT chosen will produce a crosslink only on one of the two strands of DNA, which is particularly valuable to resolve how these template strands are processed differently in a blocked replication fork. By molecular and genetic analysis, the project will validate that the engineered constructs produce DNA-protein crosslinks, block replication and determine if they promote genetic instability. Mutants in various repair pathways will be assayed for their roles in the process.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.