Defects in many genes with roles in DNA break repair are associated with a striking predisposition to cancer development. One of the most extreme cancer risks is associated with Bloom syndrome (BS) - a chromosome breakage disorder caused by mutations in the RecQ-like DNA helicase BLM. RecQ-like helicases and their role in regulating recombinational DNA repair are conserved from bacteria to humans. Besides BS, defects in RecQ-related genes cause Werner syndrome and Rothmund-Thompson syndrome, which are characterized by accelerated aging and/or increased cancer risk. In addition to BS-associated mutations, 93 missense mutations in the human BLM gene have been reported, but it is unknown which, if any, affect BLM function. It has also been suggested that single nucleotide polymorphisms (SNPs) in introns of BLM that have been associated with higher cancer risk may be linked to coding SNPs in exons of BLM. Using a yeast Sgs1-BLM chimera, we have identified coding SNPs that impair BLM function. They include hypomorphic mutations that define a new class of BLM alleles, not associated with BS, that may increase genome instability, cancer risk and other BS-associated symptoms. One objective of this proposal therefore is to determine the effect of coding SNPs throughout the BLM gene on chromosome stability, DNA break repair and the DNA-damage response, and identify their biochemical defects. In contrast to the helicase core, the >600-residue long N- terminal tails of BLM and the related yeast helicase Sgs1 are disordered and not conserved at the sequence level. They have therefore been refractory to conventional approaches to elucidate their function. It is our hypothesis that the function of the long tails of Sgs1 and BLM arises from structural elements, embedded in disorder, that serve as molecular recognition elements for binding proteins. To test this hypothesis we have designed an approach that combines computational prediction of disorder and interactivity, structure analysis by nuclear magnetic resonance (NMR) spectroscopy, and proline mutagenesis to identify these structural elements and elucidate their importance for BLM and Sgs1 function. Specifically we will (1) use a population- based mutational approach to identify and characterize novel functional motifs in BLM; the ability of BLM variants to rescue high sister-chromatid exchange, double-strand-break-repair defects and hypersensitivity to DNA-damaging agents will be assessed; (2) identify biochemical defects of functionally impaired BLM variants by assessing ATPase, DNA binding, annealing and unwinding activities, and (3) determine disorder-function relationships in the N-terminal tails of Sgs1 and BLM using a combination of (a) NMR to identify regions that are dynamically constrained and may adopt interaction-prone a-helices, (b) proline mutagenesis to disrupt the structural motifs, and (c) functional analysis of novel separation-of-function alleles of SGS1 and BLM in vivo. New insights into function and connectivity of BLM and Sgs1 will elucidate the mechanisms of hyper- recombination and chromosome instability in Bloom syndrome and, generally, in human cancers.

Public Health Relevance

The molecular mechanism underlying chromosome instability - a hallmark of most human cancers - are still unclear. The proposed studies will provide functional insights into the role of the DNA unwinding enzymes BLM and Sgs1 in the maintenance of genome integrity and will shed light on the general mechanisms leading to chromosomal rearrangements, which are often associated with human cancers and heritable chromosome breakage syndromes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081425-08
Application #
9276693
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Willis, Kristine Amalee
Project Start
2008-08-01
Project End
2019-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
8
Fiscal Year
2017
Total Cost
$292,165
Indirect Cost
$92,165
Name
University of South Florida
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
Campos-Doerfler, Lillian; Syed, Salahuddin; Schmidt, Kristina H (2018) Sgs1 Binding to Rad51 Stimulates Homology-Directed DNA Repair in Saccharomyces cerevisiae. Genetics 208:125-138
Arora, Sucheta; Deshpande, Rajashree A; Budd, Martin et al. (2017) Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast. Mol Cell Biol 37:
Shastri, Vivek M; Schmidt, Kristina H (2016) Cellular defects caused by hypomorphic variants of the Bloom syndrome helicase gene BLM. Mol Genet Genomic Med 4:106-19
Kennedy, Jessica A; Syed, Salahuddin; Schmidt, Kristina H (2015) Structural Motifs Critical for In Vivo Function and Stability of the RecQ-Mediated Genome Instability Protein Rmi1. PLoS One 10:e0145466
Bastos de Oliveira, Francisco Meirelles; Kim, Dongsung; Cussiol, José Renato et al. (2015) Phosphoproteomics reveals distinct modes of Mec1/ATR signaling during DNA replication. Mol Cell 57:1124-1132
Doerfler, Lillian; Schmidt, Kristina H (2014) Exo1 phosphorylation status controls the hydroxyurea sensitivity of cells lacking the Pol32 subunit of DNA polymerases delta and zeta. DNA Repair (Amst) 24:26-36
Kennedy, Jessica A; Daughdrill, Gary W; Schmidt, Kristina H (2013) A transient ?-helical molecular recognition element in the disordered N-terminus of the Sgs1 helicase is critical for chromosome stability and binding of Top3/Rmi1. Nucleic Acids Res 41:10215-27
Mirzaei, Hamed; Schmidt, Kristina H (2012) Non-Bloom syndrome-associated partial and total loss-of-function variants of BLM helicase. Proc Natl Acad Sci U S A 109:19357-62
Doerfler, Lillian; Harris, Lorena; Viebranz, Emilie et al. (2011) Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability. Genome Integr 2:8
Mirzaei, Hamed; Syed, Salahuddin; Kennedy, Jessica et al. (2011) Sgs1 truncations induce genome rearrangements but suppress detrimental effects of BLM overexpression in Saccharomyces cerevisiae. J Mol Biol 405:877-91

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