NIST researchers develop a novel approach for the measurement of a crucial DNA repair enzyme in human cells

Jan 08, 2014 by Miral Dizdar

(Phys.org) —NIST researchers recently reported a novel approach for identifying and quantifying an important DNA repair enzyme in mammals including humans. This enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), plays a critical role in base excision repair of DNA damage. APE1 plays a key role in repairing mutagenic base-free sites in DNA by first generating strand breaks which paves the way for other enzymes to fully repair DNA.

It has been shown that mice with a deleted gene coding for APE1 cannot survive, underscoring the critical nature of this enzyme. APE1 also plays an important role in dictating DNA repair capacity, which can determine how patients respond to cancer therapies. High expression of APE1 has been associated with resistance to therapy for many cancers. Mounting evidence points to the predictive and prognostic value of APE1 expression and subcellular localization in cancers. APE1 typically associates with a good prognosis, whereas combined cytoplasmic and nuclear localization correlates with poor survival. Moreover, mutations to APE1 are thought to dispose individuals to cancer.

Therefore, accurate measurements of APE1 levels in human tissues are essential for evaluating APE1 as a predictive and prognostic biomarker in cancer and guiding cancer treatments. Researchers in NIST's Material Measurement Laboratory developed a novel approach for the positive identification and absolute quantification of APE1 in human cells using liquid chromatography-isotope-dilution tandem mass spectrometry (LC-MS/MS). Researchers first expressed and purified a full-length human isotopically-labeled APE1 from genetically engineered E. coli cells, which was used as an internal standard for MS measurements.

Hydrolysis of APE1 and 15N-labeled APE1 with a proteolytic enzyme and subsequent analysis by LC-MS/MS resulted in identification of numerous peptides. Subsequently, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human normal and cancer cell lines. Nuclear extracts showed greater levels of APE1 than cytoplasmic extracts and cancer cells exhibited greater expression level of APE1 than normal cells. The same approach was also used to measure APE1 in mouse tissue, and APE1 variants found in humans. Overall, this work is an important first step for understanding the role of this enzyme in disease and therapeutic efficacy. Future studies will explore the broader utility of this method for measuring APE1 in patient samples.

This work has recently been published in the journal PloS One.

Explore further: Devices designed to identify pathogens in food

More information: Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Mengxia Li, M., Wilson III, D. M., Dizdaroglu, M. "Identification and quantification of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in human cells by liquid chromatography/isotope-dilution tandem mass spectrometry," PLoS One 8(7), e69894, 2013.

Related Stories

Genetic mutation causes lupus in mice

Jan 03, 2014

Yale researchers have identified a genetic mutation that leads to lupus in mice. The discovery could open the way for development of therapies that target the mutation. The study appears in Cell Reports.

Mechanism of damaged DNA mutation identified

Jan 07, 2014

A team led by KAIST Department of Biological Sciences' Professor Kwang-wook Choi and Dr. Seong-tae Hong has successfully investigated the operational mechanism of the protein ATM (Ataxia telangiectasia mutated), ...

Recommended for you

Research center develops single-cell analyzer

13 hours ago

Researchers at Missouri University of Science and Technology have developed a probe capable of detecting signs of disease or environmental change inside a single human cell.

Devices designed to identify pathogens in food

May 27, 2015

Researchers at the National Polytechnic Institute (IPN) in Mexico have developed a technology capable of identifying pathogens in food and beverages. This technique could work in the restaurant industry as ...

Biosensor may improve clinical diagnosis of influenza A

May 27, 2015

Sensors based on special sound waves known as surface acoustic waves (SAWs) are capable of detecting tiny amounts of antigens of Influenza A viruses. Developed by A*STAR researchers, the biosensors have the ...

New chip makes testing for antibiotic-resistant bacteria faster, easier

May 26, 2015

We live in fear of 'superbugs': infectious bacteria that don't respond to treatment by antibiotics, and can turn a routine hospital stay into a nightmare. A 2015 Health Canada report estimates that superbugs have already cost Canadians $1 billion, and are a "serious and growing issue." Each year two million people in the U.S. contract antibiotic-re ...

User comments : 0

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.