DrAlexander Sobinoff

Project Opportunities

Title: Unraveling the role of DBHS Proteins in genome stability and DNA repair pathway choice.

Summary of opportunity:

This project investigates the role of DBHS proteins in maintaining genome stability, with a focus on their function in resolving DNA secondary structures and influencing DNA repair pathway choice. Using a suite of molecular tools, including knockout cell lines and small molecule inhibitors, we aim to uncover the mechanisms by which these proteins regulate telomere maintenance and cellular responses to genotoxic stress.

Opportunity synopsis:

The Drosophila Behaviour/Human Splicing (DBHS) protein family comprises a group of highly conserved RNA/DNA-binding proteins that play pivotal roles in RNA processing and genome maintenance. Notably, DBHS proteins are key regulators in resolving R-loop structures and facilitating non-homologous end joining (NHEJ) in response to ionising radiation. They also bind to G-quadruplex (G4) secondary structures found in long non-coding RNAs and gene promoter regions, structures that can impact transcription and genome stability.

Despite these important associations, the underlying molecular mechanisms by which DBHS proteins mediate these functions remain poorly defined. To address this knowledge gap, we have developed a comprehensive molecular toolkit that includes:

•          siRNA-resistant mutant constructs for each DBHS family member

•          CRISPR/Cas9-engineered knockout cell lines in cancer and induced pluripotent stem (iPS) cells

•          Selective small-molecule inhibitors targeting DBHS proteins

These tools provide a unique opportunity to dissect the molecular function of DBHS proteins in maintaining genome integrity and regulating DNA repair.

Project Aims:

1.         Characterize DBHS recruitment to sites of telomeric DNA secondary structures and double-strand breaks (DSBs)

– using live-cell imaging, chromatin immunoprecipitation, and advanced microscopy techniques.

2.         Determine the impact of DBHS protein depletion on alternative lengthening of telomeres (ALT) phenotypes and DNA repair pathway choice

– including detailed analysis of NHEJ and homologous recombination dynamics in knockout cell lines.

3.         Evaluate the effects of pharmacological NONO inhibition on telomere replication stress and cellular radiation resistance

– to assess the therapeutic potential of DBHS inhibition in sensitising cancer cells to genotoxic stress.

Significance:

This project will provide novel insights into how DBHS proteins regulate genome stability under physiological and stress conditions, with broader implications for cancer biology, telomere maintenance, and therapeutic DNA damage response modulation.

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Project Opportunities

Title: Mechanisms and therapeutic targeting of anaphase DNA bridge resolution in cancer cells.

Summary of opportunity:

This project explores a newly identified DNA repair event that occurs on anaphase bridges just before cell division involving DNA synthesis at the midbody. By defining the conditions and proteins involved, it aims to uncover a potential therapeutic vulnerability in cancer cells.

Opportunity synopsis:

During mitosis, unresolved replication intermediates can give rise to DNA bridges, either chromatin bridges or ultra-fine bridges (UFBs), that physically link segregating chromosomes during anaphase. Failure to resolve these structures compromises genome integrity, resulting in mechanical DNA breakage and the formation of chromosomal rearrangements, translocations, and telomere fusions. While the resolution of anaphase bridges is essential for faithful cell division, the underlying repair processes remain poorly defined and are rarely targeted in cancer therapies.

Our preliminary data have uncovered a previously uncharacterised DNA repair event involving localized DNA synthesis at anaphase bridges near the midbody immediately prior to cytokinesis. This project will investigate the cellular conditions that trigger this repair mechanism, identify the molecular players involved, and assess whether interfering with this process, particularly in the context of replication stress, can be exploited as a novel therapeutic strategy to selectively target cancer cells.

----------Current Research Projects----------

_x000D_ 1. Mechanistic Roles of DBHS Proteins in DNA Replication and Repair

_x000D_ The Drosophila Behaviour/Human Splicing (DBHS) protein family are a group of highly conserved RNA/DNA-binding proteins implicated in multiple aspects of RNA processing and genome maintenance. These proteins are particularly important for the resolution of R-loops and promote non-homologous end joining (NHEJ) following ionising radiation. In addition, they interact with G-quadruplex (G4) structures in long non-coding RNAs and promoter regions. Despite these associations, the precise molecular mechanisms underlying DBHS function in these contexts remain poorly understood.

_x000D_ To address this gap, we have developed a comprehensive molecular toolkit, including siRNA-resistant mutant constructs for each DBHS member, knockout cancer and iPS cell lines, and DBHS small molecule inhibitors. These tools will enable detailed mechanistic studies of how DBHS proteins resolve DNA secondary structures and influence DNA repair pathway choice following exogenous stress.

_x000D_ 2. Mechanisms of Anaphase Bridge Resolution

_x000D_ During mitosis, unresolved replication intermediates can give rise to DNA bridges, classified as chromatin bridges or ultra-fine bridges (UFBs), that span segregating chromosomes in anaphase. Failure to resolve these structures can result in mechanical breakage, leading to chromosomal rearrangements, translocations, and fusions. Targeting the resolution of anaphase bridges, particularly in combination with agents that induce replication stress, represents a promising but underexplored strategy for cancer therapy.

_x000D_ Preliminary data from our lab indicate a novel, previously uncharacterised repair process involving DNA synthesis on anaphase bridges just prior to cell division. This project aims to define and characterise this previously undescribed repair mechanism.

_x000D_ 3. DBHS Proteins in Telomerase Recruitment and Telomere Maintenance

_x000D_ Telomerase, a ribonucleoprotein complex, is essential for maintaining telomeric repeat sequences at chromosome ends in proliferative cells and is a critical regulator of cellular lifespan. Its dysregulation is implicated in a broad spectrum of human diseases, from premature aging syndromes to cancer.

_x000D_ Our recent findings reveal that telomerase-mediated telomere extension in cancer cells requires DBHS family of proteins (NONO, SFPQ, and PSPC1). We are now expanding this work to investigate the role of these proteins in telomerase recruitment and function within stem cells, aiming to elucidate conserved mechanisms of telomerase regulation.