A bacterial virus protein called Gam fused to green fluorescent protein “lights up” dangerous double-stranded breaks in DNA, giving scientists a new tool with which to quantify this problem that can destabilize the genome, driving both evolution and cancer, said researchers led by those from Baylor College of Medicine in a report that appears online in the open access journal eLife.
“This new tool is important because it enables us to quantify how often these breaks occur in ways not possible before. It has already let us learn new things about how DNA breaks occur in bacterial, mouse and human cells,” said Dr. Susan Rosenberg, professor of molecular and human genetics at BCM and corresponding author of the report. Drs. Kyle M. Miller of The University of Texas at Austin, and Reuben S. Harris, of the University of Minnesota are also corresponding authors.
Green fluorescent protein
The protein Gam was chosen because it binds only to double-strand breaks in DNA, trapping them. It does not bind to other proteins or unbroken DNA. When the fused protein GamGFP is introduced into cells by genetic engineering techniques, the sites of the double-strand breaks literally light up, via the green fluorescent protein.
Rosenberg and her colleagues used the new tool to determine how spontaneous double stranded breaks occur in both bacterial and mammalian cells, estimating that the GamGFP (the fused protein) detects between 71 percent and 82 percent of double-strand breaks in bacteria.
Their findings also support the notion that many spontaneous double-strand breaks in DNA occur during replication, when the DNA duplicates itself during cell division. They also found evidence for a type of DNA breakage that involves the innate immune system and is specific to primates (including humans).
In an accompanying perspective, Dr. Michael Cox of the department of biochemistry at the University of Wisconsin-Madison mentioned some limitations to the technique in certain situations noted by the authors, but stated “this new technology is destined for creative application to important problems in eukaryotic cell biology. The list of potential experiments seems endless.”
The new technique is part of the development of innovative new tools for biology, spearheaded by the Rosenberg lab, to study proteins that cause DNA damage and may promote cancer, supported by a National Institutes of Health Director’s Pioneer Award. These special awards allow pioneering researchers to take risks for problems of high impact in biomedical research. The Rosenberg lab is discovering new proteins that cause DNA damage, and may instigate cancer, and making new tools to study them.
Others who took part in this work include: Chandan Shee, Franklin Gu, Mohan C Joshi, David Magnan, Jennifer A Halliday, Ryan L Frisch, Janet L Gibson, Ralf Bernd Nehring, Marcos Hernandez, Christophe Herman, PJ Hastings and David Bates, all of BCM; Ben D Cox and Li-Ya Chiu, both of UT Austin; Elizabeth M Luengas and Reuben S Harris of the University of Minnesota in Minneapolis; and Huong G Do and Lei Li, both of UT MD Anderson Cancer Center.
Funding for this work came from: the National Institute of Health Director’s Pioneer Award (DP1-CA174424 Susan M Rosenberg); the Cancer Prevention Research Institute of Texas (R1116 Kyle M Miller); National Institutes of Health (R01-GM53158 Susan M Rosenberg);National Institutes of Health (F32-GM095267 Ryan L Frisch); National Institutes of Health (CA127945 and CA097175 Lei Li); National Institutes of Health (R01-GM88653 Christophe Herman); National Institutes of Health (R01-GM102679 David Bates); National Institutes of Health (R01-AI064046 and P01-GM091743 Reuben S Harris).
Rosenberg holds the Ben F. Love Chair in Cancer Research and is a member of the NCI-designated Dan L Duncan Cancer Center at Baylor College of Medicine.