The orange wheel shows the circular chromosome or genome of E. coli bacteria. The spikes indicate where a molecular intermediate in DNA repair—four-way structures called Holliday junctions—accumulate near a repairable double-strand break in the genome. Holliday junctions, it has been demonstrated, are subject to trapping, mapping, and quantification by engineered proteins. [Jun Xia and Qian Mei/Baylor College of Medicine]
Fleeting expressions and gestures can be more revealing than static poses, which is why candid snapshots are often valued more than formal portraits. Yet snapshots of cancer are few and far between. Cancer, in its earliest stages, is unusually camera-shy, biochemically speaking. When cancer originates in DNA repair mechanisms that turn awry, tell-tale DNA repair intermediates disappear quickly, eluding capture by ordinary means and frustrating would-be scientific paparazzi.
To take the most candid images of cancer’s origins, scientists based at Rice University and the Baylor College of Medicine decided to exploit synthetic biology. They engineered proteins capable of capturing short-lived intermediates of DNA repair. Between the original, damaged DNA and the final, repaired product, cells produce DNA reaction intermediates, which are crucial to DNA repair but are difficult to study because they are present for just a fraction of a second as an enzyme catalyzes the changing of one molecule into another.
“The intermediate molecules are the most important parts of biochemical reactions,” said Susan Rosenberg, the leader of the Cancer Evolvability Program at Baylor’s Dan L. Duncan Comprehensive Cancer Center. “They define what the reaction is and how it will proceed. But because they are transient and elusive, it's really difficult to study them, especially in living cells. We wanted to do that. We decided to invent synthetic proteins that would trap DNA reaction intermediates in living cells.”
Dr. Rosenberg led a study (“Holliday Junction Trap Shows How Cells Use Recombination and a Junction-Guardian Role of RecQ Helicase”) that appeared November 18 in the journal Science Advances. In this study, engineered proteins were used to trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in a DNA repair mechanism called homologous recombination (HR). The engineered proteins were based on catalytically deficient mutant RuvC protein of Escherichia coli.
“We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome,” wrote the authors of the Science Advances study. “Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication.”
When cells divide and make copies of the instructions encoded in their DNA, the DNA unwinds and becomes vulnerable to damage that must be repaired. Sometimes the process of repairing the DNA can also cause mutations and errors. When these errors accumulate, the cells may acquire characteristics of cancer.
“We want to use synthetic proteins to study mechanisms that change DNA sequence,” Rosenberg said. “We do that now with genetics and genomics in my lab. But genomics, which allows us to compare the genes of normal cells with those of cancerous cells, is like reading the fossil record of these processes. We want to see how the real-time processes that change DNA happen, including all the intermediate steps, which our synthetic proteins allow us to freeze in time and isolate.”
In their tests, Rosenberg and colleagues found they could discover molecular mechanisms underlying genome instability, a hallmark of cancer. In one instance, they discovered a new role for a protein that is related to five human cancer proteins.
“We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR–HJs promoted by RecA overproduction,” the study’s authors detailed. “We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers.”
Rosenberg and colleagues think that their approach offers significant advantages. For instance, with the synthetic proteins, they have been able to identify specific DNA-repair intermediate molecules, their numbers in cells, rates of formation, and locations in the genome and the molecular reactions in which they participate.
“When you know these reactions, as well as the roles that the intermediates play in the mechanisms that change DNA, you can think about making drugs that will stop them,” Rosenberg noted. “In the future, we hope we will be able to design drugs that target specific types of cancers—drugs that block the cells' ability to evolve into cancer cells, instead of, or in addition to, traditional chemotherapies that kill or stop cancer cells from growing.”