Doctor of Philosophy (Ph.D.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Kristina H. Schmidt, Ph.D.
Alvaro Monteiro, Ph.D.
Meera Nanjundan, Ph.D.
Sandy Westerheide, Ph.D.
Bloom syndrome, DNA repair, DNA replication, Genome instability
Genomic instability is a hallmark of disorders in which DNA replication and repair genes are dysfunctional. The tumor suppressor RECQ helicase gene BLM encodes the 3’-5’ DNA Bloom syndrome helicase BLM, which plays important roles during DNA replication, recombination and repair to maintain genome stability. Mutations within BLM cause Bloom syndrome, an autosomal recessive disorder characterized by growth defects, immunodeficiency, >10-fold higher sister chromatid exchange compared to normal cells, and an increased predisposition to a wide range of cancers from an early age. Single nucleotide polymorphisms or SNPs in BLM have been reported to be associated with susceptibility to a variety of malignancies. However, these are non-coding SNPs with no evidence linking them to SNPs in BLM exons. An overwhelming majority of nonsynonymous SNPs in BLM for which allelic frequencies are known are rare. Together with their rarity in the human genome and a higher likelihood of being functional, rare SNPs can be an important genetic basis of common human diseases. And since almost all nonsynonymous SNPs in BLM are rare, they are prime candidates for functional characterization to determine risk-association. A comprehensive functional evaluation of coding BLM variants using a humanized yeast model identified six variants which totally impair BLM function, with DNA damage sensitivity conferred by these alleles comparable to Bloom syndrome-causing alleles in the human population. An intermediate sensitivity to DNA damage was conferred by three other alleles. Here, functionally evaluated the ability of 8 of these BLM variants to complement cellular defects in the Bloom syndrome patient-derived GM08505 cell line including the elevated sister-chromatid exchanges frequency, delayed response to DNA damage, and hypersenstivity to DNA damaging agents. We determined that five of these variants are incapable of complementing defects of the GM08505 cell line, which are candidates for adding to the list of thirteen currently known Bloom syndrome causing missense mutations. We identified three other BLM variants that were incapable of complementing the elevated sister-chromatid exchange frequency and delayed DNA-damage response, but were able to suppress hypersensitivity to DNA damaging agents as well as wildtype BLM. These intermediate BLM variants are hypomorphic which, instead of causing Bloom syndrome, may confer increased cancer predisposition or lead to other Bloom‐syndrome‐associated disorders like type‐2 diabetes.
The GM08505 cells used in the functional characterization of BLM variants is aneuploid. Defects in DNA replication, telomere maintenance, chromosomal segregation, cell cycle checkpoint activation, and DNA damage response cause chromosomal instability. Aneuploidy is considered to be an outcome of chromosomal instability. Paradoxically, another school of thought considers aneuploidy as the driver of chromosomal instability. It has been suggested that variable expression of genes on aneuploid chromosomes can potentially dysfunction of their protein product, leading to chromosomal instability phenotypes. It has been proposed that chromosomal instability and aneuploidy are part of a vicious cycle which results is an increasingly diverse karyotype directly impacting gene expression. Using genome engineering, we generated and functionally characterized a diploid BLM knockout cell line. This cell line exhibits cellular defects similar to the Bloom syndrome patient derived GM08505 cell line. Being diploid and unaffected by uneven gene dosage, this cell line can provide a good system to investigate dysregulated factors in the absence of BLM.
The prevention of genomic instability depends on multiple pathways ensuring timely progression of replication and appropriate response to DNA damage. BLM functions at the crossroads of response pathways induced by DNA damage during replication. The canonical role of BLM is dissolving double Holliday junction during homologous recombination- mediated DNA double strand repair as part of the BLM/Topoisomerase IIIα /RMI1/RMI2 or BTR complex to yield non-crossover products. Additionally, BLM functions in the key early step of resecting double strand breaks to initiate DNA repair. BLM can also reverse the invasion of the resultant single stranded 3’ overhangs in an anti-reombinogenic role and regulate recombinational DNA repair. BLM functions to restart stalled replication forks resulting from DNA damage. Thus far, most studies evaluating the role of BLM in replication have proposed models based on its role in recovery from induced replication stress. However, delayed replication timing, increased replication fork pausing, endogenous DNA damage, and activation of replication checkpoints in unperturbed Bloom-syndrome cells point to a role for BLM in protecting the replisome movement through the genome. A critical aspect of BLM functionality is its substrate specificity to a variety of DNA structures. Notably, it can bind and unwind G-quadruplex structures in vitro and has been associated with a functional role in unwinding such regions in vivo to aid replisome movement with limited evidence. We immunoprecipitated BLM in mid-S-phase and performed a mass spectrometric screen to identify novel interactors in a bid to identify mechanisms by which BLM maintains genome stability during unperturbed replication. We show that Mcm6, an MCM replicative helicase subunit, is a novel BLM interactor. BLM interacts with Mcm6 via multiple, distinct binding sites in G1 phase, at active replisomes in S-phase, and during response to replication stress induced DNA damage. We show that BLM is a constitutive component of active replisomes in during unperturbed replication. Finally, we uncover a potential novel role for BLM in G1 requiring interaction with Mcm6.
Our data provides functional and mechanistic insight into polymorphisms in BLM, and their implications for the general population. We have also engineered a diploid BLM knockout cell line which can serve as a platform for investigating BLM variants in the absence of uneven gene dosage as well as proteomic analysis to identify factors dysregulated in the absence of BLM. We show that BLM is a component of active replisomes in an unperturbed S phase and has a potentially novel function in G1.
Scholar Commons Citation
Shastri, Vivek M., "Novel Insights into the Multifaceted Roles of BLM in the Maintenance of Genome Stability" (2019). Graduate Theses and Dissertations.