New technology precisely measures DNA breaks in vivo: an interview with CCR’s Andre Nussenzweig

Dr. Andre Nussenzweig is an NIH Distinguished Investigator and Chief of the Laboratory of Genome Integrity at the NCI’s Center for Cancer Research.

Genome instability is a hallmark feature of cancer.  Andre Nussenzweig, Ph.D.’s lab focuses on understanding the consequences of genome instability and the application of these basic research insights into the development of new technologies and strategies for cancer prevention and therapy.

In this interview, Dr. Nussenzweig answers questions on his latest study, which was published in Molecular Cell.

What is the most important finding of your study: DNA breaks and end-resection measured genome wide by end sequencing (End-seq); why is this so?

Whenever there is an important technological advance, there is a great potential to gain new biological insights. For example, in 1998, my first year at NIH, Dr. William Bonner discovered that a histone variant H2AX—a marker for DNA damage—is phosphorylated over a large region surrounding a double strand break site; this allowed for the visualization of DNA breaks using an antibody raised against the phosphorylated form of H2AX. For the last 18 years, the science community has used this as the main methodology to monitor genome integrity. Our new technology goes one step further in resolution because it tells us with great precision exactly where the break is, and how it has been processed.

Did this study build on your lab’s previous work?

It certainly did build on our previous work. We were fortunate to collaborate extensively with Dr. Bonner, and developing END-seq forced me to re-read and re-evaluate some of these classic papers.

What is your next step?

We plan to apply this technology to new biological questions. For example, recent studies suggest that physiological brain activity “just thinking” causes DNA double strand breaks in neurons. We want to know where these breaks are, what causes them, which enzymes repairs them, and what is the consequences of mis-repair for brain function, physiology and pathology, including brain tumors.

How was this study conducted and what challenges did you and your team encounter and/or overcome?

When a research fellow, Dr. Andres Canela, joined the lab, I told him that the field would benefit if we developed a precise method to monitor DNA breaks. Andres, who is a molecular biologist, took up the challenge. Together with a new Ph.D. student, Nicholas Sciascia and a two postdoctoral fellows, Drs. Sriram Sridharan and Anthony Tubbs, it took several years to develop the method and analysis pipeline. The main difficulty with any precision measurement is that your signal is competing with “noise.” By decreasing the number of steps involved in the method, Andres and his colleagues finally succeeded in producing the most sensitive method for monitoring DNA breaks to date. Conservative estimates indicate that END-seq is at least 100x more sensitive than similar techniques currently in use.

How did efforts of your colleagues benefit this study?

We were fortunate to obtain intramural funding (CCR-FLEX award) to support this study. Additionally, we worked very closely with Dr. Paul Meltzer’s group (Genetics Branch, NCI) to sequence the DNA breaks identified by End-seq. We continue to collaborate with Dr. Mirit Aladjem (Developmental Therapeutics Branch, NCI) on understanding how DNA breaks are generated during DNA replication stress.

How has the scientific community received your work?

I am pleased that the scientific community has received this new technology with great enthusiasm, and we have started various collaborations with intramural and extramural investigators who are interested in measuring the location and frequency of DNA breaks. Since DNA breaks have the potential to destabilize the genome, being able to monitor them accurately is a fundamental advance for the Laboratory of Genome Integrity and the cancer research community.

Summary Posted: 07/2016