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Principal Investigator: NIKOLA P PAVLETICH
Organization: SLOAN-KETTERING INST CAN RESEARCH
Fiscal Year: 2024
Award: $672,595
Funding agency: National Institute of General Medical Sciences
Genetic instability is a hallmark of cancer. The cell has evolved an intricate set of pathways that sense and
repair DNA damage, which is an inevitable consequence of cellular metabolism and the environment. Failure
of a repair pathway, due to either overwhelming DNA damage or pathway inactivation by somatic or inherited
mutations, can lead to the propagation of genetic errors that confer a selective advantage to the cell and drive
the development of cancer. Among the many DNA repair pathways, those that respond to lesions on both
strands of the DNA are particularly important, as their failure can lead to chromosomal instability that can
accelerate the loss of tumor suppressor genes and the amplification of oncogenes. Such lesions include DNA
double strand breaks (DSBs) and stalled replication forks, which are DNA structures arising during the
duplication of the genome. The objective of this proposal is to understand how the cell senses DSBs and
stalled forks, and how it triggers a response with wide-ranging effects that include arrest of cell growth and
initiation of repair programs. We plan to use the method of cryo-electron microscopy (cryo-EM) to determine
the 3-dimensional structures of protein assemblies involved in these processes. Structural information –
essentially detailed images – will help us better understand how these pathways work, how they fail in cancer,
and may ultimately help identify new approaches to intervene therapeutically. Central to the sensing of a
stalled replication fork is the ATR protein kinase that signals to other proteins by phosphorylating them. ATR
and its partner ATRIP sense persistent single-stranded DNA (ssDNA) and a dsDNA-ssDNA junction – two
defining features of a stalled fork. The ssDNA is coated by the replication protein RPA, which recruits ATR-
ATRIP. The dsDNA-ssDNA junction is sensed by another protein complex that loads a clamp, termed 9-1-1,
onto dsDNA. 9-1-1 then recruits the TopBP1 protein, which binds to ATR-ATRIP and turns on the
phosphorylation activity. This is one assembly, reconstituted from purified proteins, that we plan to investigate
with cryo-EM. We also plan to investigate a related assembly, where TopBP1 is replaced with the ETAA1
protein, and which senses different features of a stalled fork. Another aspect we plan to investigate is the
remodeling of the stalled fork to facilitate its sensing and repair, and its protection during this process. These
functions are carried out by 12 FANC proteins mutated in the inherited Fanconi Anemia Cancer predisposition
syndrome. FANCM remodels the fork and recruits a 9-protein complex (FA Core complex) that puts a clamp
consisting of FANCI and FANCD2 onto the DNA, likely to protect the fork. The sensing of DSBs is mediated by
ATM, protein kinase mutated in the cancer syndrome Ataxia-Telangiectasia. DSB ends, together with a 3-
protein complex termed MRN, activate ATM and initiate the DSB response. Our third major goal is to
understand how this process works at the level of 3-dimensional structure.
Terms: <3-D structure><3-dimensional structure><3D structure><ATM Protein><ATM Serine/Threonine Protein Kinase><ATM activation><ATM kinase><ATM protein kinase><ATP phosphohydrolase><ATP-protein phosphotransferase><ATPase><ATR gene><ATR protein><ATR protein kinase><ATR serine/threonine kinase><Acceleration><Address><Adenosine Triphosphatase><Amino Acids><Anti-Oncogenes><Antioncogenes><Ataxia Telangiectasia><Ataxia Telangiectasia Mutated><Ataxia Telangiectasia Protein><Ataxia Telangiectasia Syndrome><Ataxia Telangiectasia and Rad3 Related Protein><Ataxia Telangiectasia and Rad3-Related><Ataxia-Telangiectasia Variant 1><Ataxia-Telangiectasia and Rad3-Related Gene><Ataxia-Telangiectasia-Mutated protein kinase><Berlin Breakage Syndrome><Binding><Biochemical><Cancer Genes><Cancer Suppressor Genes><Cancer-Promoting Gene><Cancers><Cell Body><Cell Communication and Signaling><Cell Cycle Arrest><Cell Signaling><Cells><Cellular Expansion><Cellular Growth><Chromatin><Chromosomal Instability><Chromosome Instability><Clampings><Closure by clamp><Complex><Congenital Pancytopenia><Cryo-electron Microscopy><Cryoelectron Microscopy><DNA><DNA Damage><DNA Damage Repair><DNA Double Strand Break><DNA Injury><DNA Interstrand Crosslinking><DNA Repair><DNA Repair Pathway><DNA Structure><DNA lesion><DNA replication fork><Data><Deoxyribonucleic Acid><Development><Double-Stranded DNA><ETAA1><Electron Cryomicroscopy><Emerogenes><Environment><Ewing's Tumor associated antigen 1><Ewing's tumor associated antigen><FA DNA repair pathway><FA-mediated DNA repair pathway><FANCD2><FANCD2 protein><FRAP-Related Protein-1><FRP1><Failure><Family><Fanconi Anemia><Fanconi Anemia pathway><Fanconi Panmyelopathy><Fanconi anemia DNA repair pathway><Fanconi anemia complementation group D2><Fanconi anemia repair pathway><Fanconi dysplasia><Fanconi's Anemia><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Genome><Goals><Hereditary><Hybrids><Image><Inherited><Inositide Phospholipids><Inositol Phosphoglycerides><Inositol Phospholipids><Intermediary Metabolism><Intracellular Communication and Signaling><KRP protein><Kinase Family Gene><Kinases><Lesion><Louis-Bar Syndrome><MEC1><Maintenance><Malignant Neoplasms><Malignant Tumor><Mediating><Metabolic Processes><Metabolism><Methods><Modeling><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Monoubiquitination><Mutate><Mutation><Nature><Nijmegen Breakage Syndrome><Non-Polyadenylated RNA><Onco-Suppressor Genes><Oncogenes><Oncogenes-Tumor Suppressors><Ortholog><Orthologous Gene><Outcome><Pathway interactions><Peptides><Phosphatidyl Inositol><Phosphatidylinositols><Phosphoinositides><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Primary Erythroid Hypoplasia><Process><Protein Kinase><Protein Phosphorylation><Proteins><PtdIns><Publishing><RNA><RNA Gene Products><Rad3 Related Protein><Recessive Oncogenes><Ribonucleic Acid><Role><SCKL><SCKL1><Seemanova Syndrome><Seemanova syndrome 2><Signal Transduction><Signal Transduction Systems><Signaling><Single-Stranded DNA><Slide><SpKRP 85><SpKRP 95><Structure><Syndrome><Therapeutic><Transforming Genes><Transphosphorylases><Tumor Suppressing Genes><Tumor Suppressor Genes><Ubiquitin Ligase Component Gene><Ubiquitin Ligase Gene><Unscheduled DNA Synthesis><Work><Yeasts><aminoacid><ataxia telangiectasia and Rad3 related><ataxia telangiectasia mutated activation><ataxia telangiectasia mutated protein><biological signal transduction><cancer predisposition><cell growth><chromosomal breakage-immunodeficiency syndrome><conformation><conformational><conformational state><conformationally><conformations><congenital aplastic anemia><crosslink><cryo-EM><cryoEM><cryogenic electron microscopy><design><designing><developmental><ds-DNA><dsDNA><genome mutation><glycogen synthase a kinase><homologous recombination><hydroxyalkyl protein kinase><imaging><kinase-related protein><kinesin II><malignancy><neoplasm/cancer><new approaches><novel approaches><novel strategies><novel strategy><nuclease><oncosuppressor gene><pathway><phosphorylase b kinase kinase><prevent><preventing><programs><protein activation><protein complex><protein purification><protein structure><protein structures><proteins structure><reconstitute><reconstitution><recruit><repair><repaired><replication fork><replication stress><response><scaffold><scaffolding><social role><ssDNA><three dimensional structure><translocase><ubiquitin ligase>