Christelle de Renty, Ph.D.

Assistant Scientific Investigator

email: cderenty@email.arizona.edu

Phone: 520-626-3640

 

Project

Understand how BLM sumoylation regulates its function in DNA damage response and replication fork stability

Genomic instability is a major driver of tumorigenesis. Among the many repair mechanisms ensuring genome integrity, homologous recombination (HR) is a high-fidelity repair pathway. HR is involved in the repair of DNA double-strand breaks (DSBs), single-strand gaps, eroded telomeres, and in the protection of stalled or damaged replication forks.

DNA replication progression is impeded by various obstacles (damage on the DNA template, DNA-bound proteins, depletion of the nucleotide pool, etc.) that cause the replication machinery to stop (fork stalling). DNA replication checkpoint ensures that stalled forks are stabilized to promote the efficient resumption of replication once the damage has been repaired. Failure to stabilize stalled forks cause their collapse, and activation of back-up origins (dormant origins) to ensure the completion of genome duplication.

The RecQ helicase BLM, which is mutated in Bloom’s syndrome, plays multiple roles at the interface of DNA replication, repair, and recombination. Strikingly bloom’s syndrome cells are characterized by excessive HR, and an increased genomic instability, associated with high predisposition to all types of cancer. BLM’s functions in HR are regulated by post-translational modification by SUMO (small ubiquitin-related modifier). Specifically, cells expressing a BLM protein that cannot be sumoylated (SUMO-mutant BLM or SM-BLM) exhibit excess DNA damage repair foci. We hypothesize that these excess repair damage foci result from increased replication fork failure and collapse.

In order to better understand how SUMO-modification of BLM regulates its function in protecting stalled replication forks and promoting their efficient restart, we are analyzing DNA replication dynamics on single DNA molecules. 

 

Method: Analysis of DNA replication dynamics on single DNA molecules.

Newly synthesized DNA is labeled in vivo by incorporation of the nucleoside analogs iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU). DNA is prepared and microfluidic capillary channels are used to stretch and align single DNA molecules on glass slides (Sidorova et al. 2009). The stretching of the DNA is constant: 1µm/3.9kb. The incorporated IdU and CldU are visualized by immunostaining. Measurements of the replication tracks are used to calculate various parameters of replication dynamics, including replication fork velocity, fork/origin density, fork asymmetry, fork stability in presence of replicative stress, and the frequency of dormant origins firing.