Project Grant F32GM165105

PANGENOME CHARACTERIZATION OF MUTATIONAL PROCESSES AND GENOMIC INSTABILITY MECHANISMS IN BACTERIA - PROJECT SUMMARY THE RAPID EXPANSION OF COMPLETE SEQUENCED GENOMES, NOW EXCEEDING ONE MILLION HIGH-QUALITY BACTERIAL GENOME ASSEMBLIES, PRESENTS AN UNPRECEDENTED OPPORTUNITY TO UNDERSTAND THE FUNDAMENTAL MECHANISMS DRIVING MUTAGENESIS AND GENOME INSTABILITY ACROSS BACTERIAL SPECIES. CURRENT UNDERSTANDING OF HOW MUTATIONAL PROCESSES AND DNA STRUCTURE (PARTICULARLY NON-CANONICAL DNA STRUCTURES) CONTRIBUTE TO BACTERIAL GENETIC VARIATION REMAINS FRAGMENTED, LIMITING OUR ABILITY TO PREDICT AND MANIPULATE GENOME STABILITY FOR THERAPEUTIC AND BIOENGINEERING APPLICATIONS. THIS FUNDAMENTAL GAP IS PRIMARILY DUE TO THE ABSENCE OF SYSTEMATIC, LARGE-SCALE STUDIES. HARNESSING THE RAPIDLY EXPANDING REPOSITORIES OF BACTERIAL GENOME ASSEMBLIES, NOW COMPRISING OVER ONE MILLION HIGH-QUALITY GENOMES FOR HUNDREDS OF DIFFERENT SPECIES, OF DIVERSE LINEAGES AND ECOLOGICAL NICHES, WE CAN GAIN CRITICAL INSIGHTS INTO GENOMIC INSTABILITY AND POPULATION-LEVEL GENETIC DIVERSITY IN NATURE. THIS RESEARCH PROJECT WILL EMPLOY A COMPREHENSIVE COMPUTATIONAL AND EXPERIMENTAL APPROACH, LEVERAGING BACTERIAL PANGENOMES AT SCALE TO SYSTEMATICALLY CHARACTERIZE MUTATIONAL SIGNATURES AND NON-B DNA-ASSOCIATED GENOMIC INSTABILITY ACROSS HUNDREDS OF BACTERIAL SPECIES. MY FIRST HYPOTHESIS POSITS THAT MUTATIONAL SIGNATURES EXTRACTED FROM BACTERIAL PANGENOMES WILL REVEAL CONSERVED AND LINEAGE-SPECIFIC MUTATIONAL PROCESSES, DRIVING GENETIC VARIATION. I WILL VALIDATE THE INFERRED SIGNATURES BY RECAPITULATING THE MUTATIONAL PROCESSES THROUGH BACTERIAL MUTAGENESIS EXPERIMENTS FOLLOWED BY WHOLE GENOME SEQUENCING AND COMPUTATIONAL ANALYSIS. THESE COMPLEMENTARY APPROACHES BUILD UPON OUR PRELIMINARY ANALYSES OF 28,689 BACTERIAL GENOMES, WHICH SUCCESSFULLY IDENTIFIED MUTATIONAL SIGNATURES ACROSS DIVERSE BACTERIAL SPECIES. MY SECOND HYPOTHESIS STATES THAT NON-B DNA SEQUENCES SERVE AS HOTSPOTS FOR GENOMIC INSTABILITY, WITH THEIR MUTAGENIC POTENTIAL MODULATED BY ENVIRONMENTAL FACTORS AND DNA REPAIR SYSTEMS. MY PRELIMINARY ANALYSES REVEAL ELEVATED MUTATION RATES AT NON- B DNA LOCI, WITH PATTERNS VARYING BY NON-B DNA CATEGORY AND THE BACTERIAL SPECIES EXAMINED. COMPREHENSIVE MAPPING OF NON-B DNA MOTIFS AND QUANTIFICATION OF THEIR CONTRIBUTION TO GENOMIC INSTABILITY WILL BE USED TO INVESTIGATE THE INFLUENCE OF ENVIRONMENTAL FACTORS, AND DNA REPAIR PATHWAYS ON THEIR MUTAGENIC POTENTIAL. THE PROPOSED WORK IS SIGNIFICANT BECAUSE WE LACK A COMPREHENSIVE UNDERSTANDING OF THE MECHANISMS DRIVING BACTERIAL GENETIC VARIATION AND GENOME INSTABILITY. THIS RESEARCH PROJECT WILL GENERATE PRINCIPLES AND METHODOLOGIES BROADLY APPLICABLE ACROSS THE TREE OF LIFE, DEEPENING OUR UNDERSTANDING OF HOW MUTATIONAL MECHANISMS AND NON-B DNA SEQUENCES SHAPE GENETIC VARIATION IN NATURE. THESE INSIGHTS WILL DIRECTLY SUPPORT MY LONG-TERM GOAL OF DEVELOPING A COMPREHENSIVE, MECHANISTIC FRAMEWORK FOR HOW GENOME STRUCTURE AND MUTATIONAL PROCESSES DRIVE GENETIC VARIATION AND GENOME INSTABILITY.