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;

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.

In all cells, the process of genetic recombination repairs DNA damage that might otherwise lead to mutations or cell death. The loss of various DNA repair mechanisms in human cells has, in some cases, been correlated with a predisposition to cancer. Studies of genetic recombination in the bacterium Escherichia coli has defined in detail many of the proteins that mediate steps of genetic exchange. Our previous work has focused on understanding the role that DNA exonucleases play in genetic recombination in E. coli. We propose to characterize the biochemical properties of the RecJ exonuclease that is involved in several pathways for DNA repair. We will determine the properties of the RecJ exonuclease in coupled in vitro reactions with the RecA protein of E. coli. RecA plays a central role in genetic recombination and can promote strand exchange between homologous DNA molecules. We will investigate the reactions that mimic the types of biochemical steps that are thought to constitute recombinational DNA repair. These experiments will provide a biochemical framework to understand the role of RecJ in the bacterial cell. We also propose to define those regions of the RecJ protein that are important for the genetic and biochemical functions of RecJ. We will isolate and characterize mutant forms of the protein by genetic and biochemical means. We will isolate, sequence and compare the predicted amino acid sequence or other bacterial RecJ proteins to determine those residues of the protein that have been conserved throughout evolution. Our goal from this analysis will be to develop a motif shared among RecJ and other 5' exonucleases. Although 5' exonucleases are found in many genetic recombination systems, there is no great similarity among these proteins in primary sequence. It is therefore not possible to identify putative 5' DNA exonucleases based on sequence information alone. Extensive mutational analysis of exonuclease proteins has not been performed.From our proposed analysis, we will be able to identify the regions of the protein likely to be involved in catalysis; these regions are most likely to share similar structure among exonucleases. We will use the structural information about RecJ to seek RecJ-homologs from the eukaryotic organism Saccharomyces cerevisiae. Molecular genetics in the yeast S. cerevisiae is well-developed and many genes involved in genetic recombinationand DNA repair have been identified. Recently, homologs of several genes which mediate genetic recombination and DNA repair in bacteria have been found in yeast. The yeast homologs have been useful, in turn, in finding human counterparts to these genes. The conservation of DNA repair pathways from bacteria to yeast suggests that RecJ-like proteins should be found in eukaryotes. Our proposed experiments seek to identify these counterparts in yeast and to begin to investigate their genetic and biochemical properties in this eukaryotic organism.

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): 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.

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): This project supports the training of nine students annually at Brandeis University in the field of genetics, who will be appointed in the second year of their Ph. D. training. This grant has played a central role in the education of a highly productive group of interactive graduate students actively involved in genetics research in problems 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 and Biochemistry. 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 professional skills including scientific literacy, writing and oral 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 to three years. Progress of the students is closed 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. A number of professional development activities feature valuable personal discussions that assist each student's career planning. 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.

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.

This project is co-funded by the Department of Defense in partnership with the NSF REU program. The Brandeis University REU program will support the training of 10 undergraduate students for 10 weeks, during the summers of 2011-2013. Students will conduct research employing modern cell and molecular visualization techniques. A large number of faculty in the Life Sciences at Brandeis will introduce undergraduate students to a broad range of topics on biological structure and function. Training will take place in a supportive and interactive environment with state-of-the-art facilities. Participating faculty have a strong record of mentorship of undergraduates. Students will conduct full-time research guided by their mentors. In addition, they will participate in weekly lunch seminars, which will include faculty research presentations, ethics discussions, and professional development activities such as panel discussion with students and postdoctoral fellows from the Boston area on careers in biotechnology and research. Students will have an opportunity to interact with scientists, who have diverse interests and at different stages in their careers. A weekly break-out session following the seminar will feature group discussion of ethical and science issues with the speakers, in a smaller group setting. Students will develop a written synopsis of their summer work, with feedback from their mentors, and will participate in a capstone symposium including poster presentations. Students will be recruited through the web, as well as personal outreach at undergraduate research conferences and visits to local institutions. Participants will be selected based on academic record and potential for research in biology. The value of the experience for students and mentors will be measured by various assessment instruments, including a common REU assessment tool. The impact of the experience will be monitored by tracking students for continued interest in science, further educational choices, and career paths. More information can be obtained by visiting http://www.bio.brandeis.edu/undergrad/summerresearch, or by contacting the Director, Dr. Susan Lovett at lovett@brandeis.edu, or the Co-Director, Dr. Joan Press at press@brandeis.edu.

The study of mutagenesis, the theme that unites this Program Project, involves intensive analysis of DNA for the nature and frequency of mutations in particular genomic targets. This typically involves sets in the hundreds of isolates. The execution of such experiments and the analysis of data derived from these is costly and time-consuming. The determination of mutational spectra that are intrinsic to the projects demands two types of analyses: accurate DNA fragment length measurements and DNA sequencing. There is a need in the field for detection of mutations in a larger genomic context, to expand the regions that can be surveyed and to detect the types of mutations that have traditionally posed difficulties for molecular analysis, such as large genomic rearrangements. Highly parallel DNA sequencing platforms now provide an opportunity to extend the scope of mutational analysis. Common needs for the Project are DNA analyses that are convenient and high-throughput, both in physical execution and data analysis.The overall goal of this Core is to provide facilities in which state-of-the-art sequence and fragment length analysis can be coordinated, in cost-effective ways and at a scale and speed demanded by the needs of the program project investigators. A key feature of the Core will be protocoldevelopment, user training, centralized quality control, and data analysis tools, centralized on a shared Wiki site and supported through group interactions. Bioinformatic support will be provided to allow investigators to develop appropriate analysis for their unique needs. The establishment of these facilities will maximize resources, promote development and sharing of expertise and will improve the efficiency of each project.

Mutations associated with imperfect inverted repeat sequences, quasipalindromes, with potential for DNA secondary structure, are widespread. They have been noted as mutational hotspots in bacteriophage, yeast and humans and mutate by a replication template-switch mechanism. In humans, template-switch mutations contribute to a large set of genetic diseases, structural variation in genomes and to 20% of the mutations that affect p53 in human cancers. Despite the prevalence and importance of this class of mutation, little systematic work has been done to define the parameters that govern the mutagenic mechanism, mutagens specific to this class or cellular factors that influence mutation rate. The long-term goal of this study is a more complete mechanistic understanding of quasipalindrome-associated mutagenesis. Objectives will be to define the structural parameters and cellular pathways that govern mutability. Because all cells mutate and repair DNA in fundamentally similar ways by evolutionarily related pathways, these studies using the model organisms, Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster should reveal mechanisms applicable to repair of DNA in human cells. The first aim of this proposal is to investigate the connection between replication, DNA damage repair and transcription collisions with template-switch mutagenesis in E. coli. The hypothesis that targeting of exonucleases to lagging strand features via singlestrand DNA binding protein and replication clamps accounts for the observed strand bias of mutagenesis will be tested. The impact of replication fork collisions with transcription complexes will be assessed. Biochemical analysis of DNA polymerase III and its interactions with clamp and clamp-loader will be correlated with genetic effects on mutagenesis to clarify its mechanism. Additional template-switch vulnerable sites will be sought by whole-genome sequence analysis. The second aim is to develop the first eukaryotic mutational reporters for template-switch mutagenesis using the URA3 gene of S. cerevisiae. In addition, an existing lacZ reporter for mutagenesis in Drosophila melanogaster will be retrofitted to report specific types of mutations, including template-switching at quasipalindromes or direct repeats, and base substitutions and frameshifts that respond to particular types of polymerase errors or DNA damage. The effects of aging on mutation frequency will be tested using these constructs. This study will significantly advance our understanding of DNA mutagenesis by providing new tools and information about this important and neglected class of mutations.