QnAs with Susan T. Lovett

More than 50 years ago, biologists discovered that cells mount a coordinated stress response to DNA damage. When exposed to UV radiation, bacterial cells turn up processes related to DNA repair, mutagenesis, and stress tolerance. Known as the SOS response, this set of processes underlies the ability of bacteria to survive harsh environments—such as the human body—and acquire mutations that make them resistant to antibiotics. However, details of the SOS response and exactly how it is regulated remained unknown for years. Brandeis University biologist Susan Lovett uses modern techniques to study how cells repair DNA damage and regulate DNA replication. Over time, she has built an updated model of the SOS response. For her contributions to molecular biology, Lovett was elected to the National Academy of Sciences in 2021. In her inaugural Article (1), she unravels the role of the protein SspA in the cellular response to replication stress.

Susan T. Lovett. Image credit: Kevin Allen (Kevin Allen Photography, Washington, D.C.).

PNAS: How did you become interested in studying the SOS response?

Lovett: Understanding how cells recover from problems in DNA replication has been a lifelong career goal of mine. In 1967, Evelyn Witkin first proposed that all these seemingly unrelated phenomena had a common genetic control and were induced by DNA damage—that became known as the SOS response. But there is still a lot we don’t know about it. To study the SOS response, most researchers turn to things like UV radiation, ionizing radiation, or chemicals that induce very broad DNA damage across the cell. I wanted to study what happens when DNA replication has problems in the absence of other lesions outside the replication fork. To do that, I started using azidothymidine (AZT), the first approved chemotherapeutic agent for HIV/AIDS. It works against HIV because it inhibits the replication of the virus, but it turns out to also inhibit DNA polymerases in Escherichia coli. I started using the drug in E. coli in the early 2000s and showed that, when AZT blocks replication, it leads to the accumulation of single-stranded DNA gaps that elicit an SOS response. I have since done a lot of studies using AZT to look for E. coli mutants that are more sensitive or less sensitive than usual to replication stress.

PNAS: How did that work lead to your Inaugural Article?

Lovett: One of those screens for E. coli mutants turned up a gene called iraD. We reported in 2009 that mutations in this gene made cells especially sensitive to DNA damage (2). It was clear that this regulatory factor was important for cell stress responses. But what stood out to us was that neither of the canonical systems known to turn on the SOS response—LexA and RecA—seemed to have any direct effect on iraD. I was really intrigued… because I had known for years that there was a whole group of other genes that were damage-inducible through an unknown mechanism. I thought maybe this could get us to the root of that. We first identified SspA as a transcriptional activator of iraA and, in the new paper, went on to ask what the broader role of SspA in this context is.

PNAS: What did you find when you probed SspA in detail?

Lovett: To our surprise, SspA was just as important—if not more important—than the SOS response genes that we already knew about. We used RNA-Seq in bacteria treated with AZT to probe which genes are turned on in response to replication stress. Carrying out those experiments in wild-type cells, cells with a lexA mutation, and SspA knockout cells let us compare which genes are associated with which regulators. Many of the classical SOS genes’ expression is directly regulated by LexA. But quite a lot of AZT-inducible genes also have some degree of dependence on SspA; 237 genes had expression elevated more than fourfold with SspA. Many of these genes were related to nucleotide metabolism. After exposure to AZT, the increase in expression of a pyrimidine biosynthesis gene—which is likely a way for the cells to boost replication—is dependent on SspA.

The other interesting thing we saw was that the genes dependent on SspA change dramatically after AZT treatment. Some of these pyrimidine genes, for instance, are highly dependent on SspA after AZT but are actually inhibited by SspA before treatment. This suggests that there is some kind of major switch in function, but we don’t yet know the nature of that switch.

PNAS: What is the significance of studying the SOS response?

Lovett: I think there’s a notion out there that we know everything there is to know about E. coli because it has been a model organism for so long. But it’s just not true; there are still so many mysteries. There’s a lot more to learn, and there is a lot of power in applying new technology to these questions.

In bacteria, the DNA damage response is incredibly important in the evolution of pathogenesis as well as the acquisition of new toxins and of drug resistance. Superresistance mechanisms as well as persistence, where some fraction of the population can survive an antibiotic treatment, are enabled by the SOS response. When we are infected with pathogenic bacteria, our immune system reacts, and one consequence of that is DNA damage to the bacteria and the resulting SOS response. So understanding the details of this [process] could open up new avenues for developing antibiotics or fighting antibiotic resistance.

PNAS: What are your next steps in this line of research?

Lovett: The SOS response is turning out to be a lot more complex than we expected. These pathways are all interacting with each other, and there are more details to work out about their interplay. We’d like to focus on a better understanding of the switch controlling SspA-dependent genes as well as how those nucleotide biosynthesis genes and other genes not previously known to be damage-inducible are regulated by the SOS response.