Mutations and chromosome rearrangements are ubiquitous feature of cancer genomes and linked to the anomalous cellular activities associated with oncogenesis. Recombination hotspots, where genome rearrangements initiate at incongruently higher frequencies, are significantly enriched with repetitive sequence elements with the potential to fold into non-helical DNA secondary structures. When left unresolved, such DNA structures can impede transcription and replication leading to genome instability. G-quadruplex or G4 DNA is a tetra-helical structure consisting of runs of guanines and one of the DNA structures that presents a challenge to normal DNA transactions. Comprehensive understanding of how a subset of the guanine run- containing sequences interspersed in the genome becomes genetically unstable, however, is still lacking. Also not understood is how such potential hotspots of genome instability are kept under control. The long-term research goal of the applicant is to better understand how spontaneous mutagenesis and rearrangements occur at disparate rates throughout the genome. The goal of the proposed research is to characterize what potentiates the marked elevation in instability associated with certain guanine-run containing sequences in the model eukaryote, S. cerevisiae. The central hypothesis of this proposal is that genome instability at a G4 motif is contingent on both genomic context and trans-acting factors that modulate the structural transformation of a guanine-run containing sequence into G4 DNA structure. This hypothesis is founded on the preliminary data generated by the applicant that identified two critical factors in modulating instability at a model G4 DNA- forming sequence ? the level of transcription and the activity of topoisomerase I.
Our specific aims are designed to (1) define the role of topoisomerase I in the G4 DNA-associated genome instability (2) test whether DNA damage cooperatively aggravates the instability of G4 forming sequences and (3) Identify proteins binding specifically to co-transcriptionally formed G4 DNA in vivo. In combination with molecular approaches to detect G4 DNA in vivo, genetic assays developed for quantitative measurement of recombination occurring at a G4 motif in transcription-dependent manner will be employed in AIM 1 and 2 to determine the effect of disruption in Top1 function and accumulation of DNA damage on the stability of co- transcriptionally formed G4 DNA. The genetic assays have proved effective and productive in generating the relevant preliminary data.
Under AIM3, novel trans-activating factors with possible roles in suppressing instability at the G4 DNA will be identified by chromatin affinity purification approach. The stability of G4 motifs is particularly relevant to the biology of cancer since there is a significant overlap between genomic loci containing G4 sequences and the sites of translocations observed in various human cancers. The result of the proposed study can ultimately enhance our understanding of the molecular events instigating the tumorigenic rearrangements as well as therapy-related secondary malignancy or drug-resistant tumor development.
Better grasp of the mechanism of genome instability is highly relevant to public health because it is central to understanding both the changes in cells at the onset of tumorigenesis and during the treatment of cancers with chemotherapeutics that function by damaging DNA and elevating genome instability. The results from the proposed research is expected to contribute to understanding how non-canonical DNA secondary structures, namely the four-stranded G-quadruplex, reshape the genome by instigating deletion, duplication and rearrangements. By studying how genome instability is not randomly distributed but rather concentrated at certain genomic loci, this work is applicable to the part of NIH's mission to develop fundamental knowledge regarding development and treatment of cancer.
