Modification-dependent restriction systems (MDRs) recognize and cleave modified foreign DNA. These proteins are thought to play a role in establishing the epigenetic landscape of bacterial genomes and are especially important in protecting against predatory bacteriophage viruses, many of which incorporate modified bases into their DNA to evade detection by other defense systems. While MDRs can be found in mostantibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA),Clostridium difficile,andcarbapenem-resistantenterobacteriaceaelikeKlebsiellapneumoniae,noeukaryotichomologs exist,makingthempromisingtargetsfordrugdesign.Inhibitingthesesystemshasthepotentialtoenhance the efficacy of phage-mediated bacterial killing, thus providing new therapeutic strategies to combat persistent, antibiotic resistant microbial infections. It is our long-term goal to study the basic biology and mechanismsofMDRsandusethisknowledgetoimprovecurrentphagetherapyapproaches.Thisproposal examines the structure and function of the McrBC restriction system, a two-component MDR that targets DNA containing methylated cytosines. E. coli McrB contains an N-terminal DNA binding domain and a C- terminal AAA+ motor domain that hydrolyzes GTP and mediates nucleotide-dependent oligomerization. McrB?sbasalGTPaseactivityisstimulatedviainteractionwithitspartnerendonucleaseMcrC.Biochemical studies suggest a model for DNA cleavage in which McrB and McrC assemble together at two distant methylated sites and translocate in a manner dependent on stimulated GTP hydrolysis. Collision of these McrBC assemblies triggers cleavage of both DNA strands. Despite this model, the molecular and mechanisticdetailsunderlyingMcrBCfunctionremainpoorlydefined.
In Aim1, wewilldissectthespecies- specific determinants of DNA binding in different McrB homologs using X-ray crystallography and biochemistry. We will also generate chimeras that exchange the DNA binding domains between different McrB homologs to test the hypothesis that the core hydrolysis and cleavage machineries in McrBC are conservedandhaveadaptedtodifferentevolutionarypressuresviaamodulardesign.
In Aim2, wewilluse mutagenesisandkineticassaystoidentifythecriticalcatalyticcomponentsresponsibleforMcrC-stimulated GTPase activity.
In Aim 3, we will determine the structure and architectural organization of the McrBC restriction complex at atomic resolution by X-ray crystallography and cryo-electron microscopy. These effortswillprovidenewinsightsintohowMcrBCcomplexesbindDNA,assemble,andhydrolyzeGTP.

Public Health Relevance

McrBC is a conserved bacterial defense system that cleaves modified DNA. Our research objective is to understand the structure, function and regulation of McrBC at the molecular level, which may lead to new therapeutictoolsandapproachestocombatmultidrugresistantbacterialinfections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM120242-02S1
Application #
9891459
Study Section
Program Officer
Flicker, Paula F
Project Start
2018-01-01
Project End
2022-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Cornell University
Department
Other Basic Sciences
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850