Helicases are molecular motor proteins that use energy from hydrolysis of ATP to manipulate DNA and RNA in
all phases of nucleic acid metabolism. Numerous mutations have been identified in many different helicases
that are associated with human diseases including cancer, heart disease, and neurological disorders. The
primary function of helicases is to unwind duplex DNA, but other critical functions have been discovered for
which biochemical mechanisms are unknown. Helicases displace proteins from DNA and unfold secondary
structures in DNA such as G-quadruplex DNA (G4DNA) in reactions that are critical for maintaining genomic
stability. G4DNA is made of four guanines that form Hoogsteen hydrogen bonds in a planar ring which is
referred to as a G quartet. Multiple stacks of these G quartets associate to form highly stable structures.
G4DNA affects DNA metabolism including transcription, recombination, and replication. The Pif1 family of
helicases has been identified in all eukaryotes and has been identified as playing a key role in recognition and
unfolding of G4DNA structures. Mutation in Pif1 can increase the risk for some forms of breast cancer. The
overall goals of this project are to determine the mechanism(s) by which Pif1 and other helicases push proteins
from DNA and unfold critical DNA structures such as G4DNA. We will determine how helicases are affected
by proteins with which they interact such as single-stranded binding proteins and recombinases.
G4DNA sequences are found throughout the genome, but are localized preferentially to certain regions
such as promoters of proto-oncogenes, telomeres, and mitochondrial DNA. The mechanism(s) through which
these structures impart biological function are largely unknown. We have devised a method to examine the
epiproteome at practically any site in the genome by using a CRISPR-Cas9 targeting strategy. We will identify
the proteins and histone modifications that surround G4DNA sites in order to understand how these sequences
influence gene expression, recombination, and other activities. We have applied a proteomic screen to
discover new proteins that bind to G4DNA. The major proteins identified, including the RNA helicase DHX36,
are known to assemble into cytoplasmic structures termed stress granules under conditions of cellular stress.
The location of these proteins and their known roles in regulation of translation led us to test a hypothesis for
one function of G4DNA. Our data supports the conclusion that G4DNA is excised from damaged mitochondrial
and nuclear genomes and can enter the cytoplasm intact where it facilitates formation of stress granules. Our
goals now are to determine the specific sequences of G4DNA removed from the genome, the mechanism by
which the G4DNA is excised, and the specific functions by which excised G4DNA affects translation. The
long-term goal is to understand how signaling by G4DNA overlaps and intersects with other signaling pathways
such as the DNA damage response and innate immune response.