DNA

SHARING MCB SCIENCE: DISCOVERY OF A NON-BASE FLIPPING MECHANISM IN DNA REPAIR

Eichmangroup1

Eichman Lab members involved in the study (from left to right): Dr. Elwood Mullins, Dr. Brandt Eichman, Rongxin Shi, and Dr. Zachary Parsons. Photo Credit: Susan Urmy/Vanderbilt

The DNA of humans, like that of all other organisms, can be damaged, acquiring what are referred to as “lesions.” A common form of DNA damage is DNA alkylation, where a small group of carbons and hydrogens (alkyl group) are chemically bound to the base of DNA nucleotides (the As, Ts, Cs, and Gs that make up DNA). When a DNA base is alkylated, the normal function of the cell’s DNA is disrupted and the genetic information being stored is mutated, which has the potential to develop into some types of cancer and threaten the survival of the organism.

To protect the organism from the effects of DNA lesions, cells have processes to repair DNA. One such process is called base excision repair, which was one subject of last year’s Nobel Prize in Chemistry. As shown in the figure below, base excision repair begins with DNA glycosylase (ie. a protein with enzymatic function that initiates a process), which is able to bind to double-stranded DNA and look for DNA lesions using a base-flipping mechanism. In base-flipping, a DNA nucleotide that is suspected of containing an alkyl group is flipped away from its base pair partner and into the active site of the DNA glycosylase. If the DNA glycosylase sees a lesion, it severs the chemical bond that links the DNA base to the DNA backbone and initiates subsequent repair steps, ultimately restoring the DNA to an undamaged state.

Until recently, it was thought that all DNA glycosylases used base-flipping to repair damaged DNA. A paradigm shift occurred in the DNA repair field when a non-base-flipping DNA glycosylase enzyme, called AlkD, was discovered by Professor Dr. Brandt Eichman in the Department of Biological Sciences and Center for Structural Biology at Vanderbilt University and his research group, in collaboration with Professor Dr. Sheila David and her research group at University of California Davis and Professor Dr. Yasuhiro Igarashi at the Toyama Prefectural University in Japan. Repair that does not involve base-flipping has also been shown by the Eichman team to uniquely allow the repair of bulky DNA lesions.

research-summary.png

Space-filling models (left) and illustrations (right) showing base-flipping excision repair (top) and non-base-flipping excision repair (bottom). Top: A damaged DNA base (blue) from a double stranded DNA helix (orange and yellow) is inserted, or “flipped,” into the active site of the DNA glycosylase enzyme (white or grey). Bottom: A bulky chemical group (purple) attached to a DNA base (blue) results in a lesion within a double stranded DNA helix (orange and yellow) that is repaired without base-flipping by a DNA glycosylase enzyme (AlkD) (white or grey).

As described in a recent publication in Nature, the Eichman research team used a technique called X-ray crystallography to capture a series of time-lapsed 3D renderings of AlkD as it repaired a lesion. The Eichman team’s conclusion that AlkD removes DNA damage using a non-base-flipping mechanism was supported by their crystallographic analysis which showed the AlkD enzyme mainly contacted the DNA backbone, not the DNA lesion. Thus, non-base-flipping broadens the spectrum of DNA damage that DNA glycosylases are known to repair. Also, the 3D structure of AlkD is common to proteins that do not have enzymatic functions, which makes it difficult for researchers to identify non-base-flipping DNA glycosylases just based on their structure. Therefore, there is a strong possibility there are other DNA repair proteins that scientists have yet to identify.

When asked about the broader impacts of his research, Dr. Eichman responded: “This research program has involved trainees from all levels—undergraduate, graduate, and postdoctoral—several of whom have continued on in a number of scientific careers, including medical school, science policy, and industry. Most importantly, it has enabled us to expose undergraduates to cutting edge structural biology and to the practical aspects of X-ray crystallography, both in the classroom and in the lab.”

This work is funded jointly by the Genetic Mechanisms program in the Division of Molecular and Cellular Biology (MCB) and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences, Award #MCB-1122098 and Award #MCB-1517695.

Sharing MCB Science: Learning Much More about Spores

Like all living organisms, bacteria need nutrients in their environment to survive and grow. When the survival of bacteria like Bacillus subtilis is threatened by starvation they respond by going into a “hibernation” state by forming spores. The process for producing a spore, called sporulation, is highly complex and requires careful coordination with other cellular processes like DNA replication. To understand how cells are able to orchestrate this coordination, Dr. Oleg Igoshin, an Associate Professor at Rice University partnered with Associate Professor, Gurol Suel from the University of California San Diego to study sporulation of the soil bacterium Bacillus subtilis, a model organism for systems biology research.

Jatin Narula, Anna Kuchina, Dong-Yeon D. Lee, and Masaya Fujita compose the research team, led by Igoshin and Suel, interested in clarifying the genetic mechanism of spore formation or sporulation. By combining experimental methods from systems and synthetic biology with mathematical modeling, the researchers uncovered the coordination mechanism required for sporulation. The modeling predicted that the key to this coordination is the specific arrangement of two pivotal sporulation genes on the bacterial chromosome. This arrangement produces a temporary mismatch in the number of copies of these two genes during DNA replication. This mismatch is detected by the biochemical network controlling sporulation to ensure proper coordination and the completion of DNA replication. These predictions were confirmed when rearrangement of the two pivotal genes on the chromosome prevented cells from sporulating. The sporulation mechanism that Igoshin and his team elucidated is described in the video above and in a recent research article in Cell.

When asked about the broader impacts of this research for cell biology, Igoshin said “We found that the relative arrangement of the two sporulation genes on DNA were similar in more than 40 strains of spore-forming bacteria. This evidence suggests that this timing mechanism is highly conserved, and it is possible that other time-critical functions related to the cell cycle may be regulated in a similar way.”

In addition to the scientific impact of this research, the collaborative nature of the research provided interdisciplinary training for participating graduate students. Furthermore, the innovative approaches used by Igoshin and colleagues may be applicable to similar problems in other organisms and useful for teaching system-level concepts to students of various levels and backgrounds.

Jennifer Doudna featured as Influential Scientist in Time Magazine

Time Magazine recently published the “Time 100“, a list of influential leaders in their respective fields. We are pleased to report that MCB-funded investigator Jennifer Doudna was included as an influential scientist for her transformative research to develop gene editing technology.

Dr. Doudna , along with colleagues and collaborators, developed a now widely used genome editing tool known as the CRISPR-Cas1 system.  This invention emerged from Dr. Doudna’s interest in learning how an apparent bacterial adaptive immune system functions on a molecular level that is capable of protecting bacteria from deleterious foreign nucleic acids, including those delivered by bacteriophages. She and others found that CRISPR sequences represent a form of “memory” resulting from previous exposure to foreign DNAs and showed that fragments of these exogenous DNAs are integrated into the CRISPR array. Upon phage invasion, the CRISPR sequence is transcribed, together with a down-stream cas gene that encodes an endonuclease, such as Cas9 in Streptococcus pyogenes. The long, non-coding pre-CRISPR RNA (pre-crRNA) transcript is then processed, producing multiple different crRNAs. The crRNAs form a hybrid to a second CRISPR-encoded RNA called transactivating CRISPR RNA (tracrRNA), which has regions of complementarity to the various crRNAs. These RNA hybrid oligomers associate with the endonuclease and serve as a guide to target newly invading nucleic acids. Recognition of the foreign DNA triggers precise double-stranded cleavage, leading to complete nucleolytic degradation.

Understanding the molecular events by which CRISPRs function on the molecular level led Dr. Doudna and her collaborators to develop the pioneering genome editing capability that functions broadly across many species. Dr. Doudna gives an overview of this technology in the following video.

NSF funding for Dr. Doudna’s groundbreaking research began in 2007 and continues today.  Her research represents an excellent example of how fundamental research inspires innovation.

________________________________________

1CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. These repeats are often associated with coding sequences for RNA-guided DNA endonuclease enzymes, general denoted “Cas” for CRISPR-associated.

Sharing MCB Science: Watching the STARs (Small Transcription Activating RNAs)

The editors are excited about the first of what we hope to be many blog posts featuring the science of MCB-funded investigators. We plan to share a broad sampling of this research and its outcomes on our blog. If you are a) an MCB-funded researcher and b) have recently published research that you would like to share, please fill out this form to be considered for a featured post.

RNA is an important molecule found in all living organisms. To use a computer analogy, RNA acts like a circuit board, controlling DNA, nature’s hard drive. RNA helps to read genetic information and enact the programs of life, through proteins and other important biologic materials. Dr. Julius Lucks, along with post-doctoral associate James Chappell and graduate student Melissa Takahashi, has capitalized on the ability of RNA to form different folded structures via complementary base pairing to create Small Transcription Activating RNAs or STARs. STARs are a molecular “on-switch”, whose shape controls the state (off or on) of the switch. In their recent paper in Nature Chemical Biology, the Lucks Lab researchers describe using STARs to activate the first steps in gene expression (the “printing out” of proteins) in bacteria. The researchers also provide data to support the idea of snapping STARs together to create advanced genetic programs within bacteria.  These examples demonstrate the potential application of STARS to allow bioengineers or synthetic biologists to write new genetic programs, which could engineer cells to address health and environmental challenges. Ongoing research in the lab is focused on using STARs for molecular diagnostics.

The research group is committed to broader impacts and they have recently created a summer course at Cold Spring Harbor Laboratories in Synthetic Biology, where they collaborate with other synthetic biology researchers to develop an ideal training ground for students interested in learning more about the field. Students range from first-year graduate students, to industry professionals, to senior Professors. The course, which has been held for the past 3 summers, is interactive, incorporating research, top-notch guest speakers, and hands-on activities.

To engage the broader community in synthetic biology, the Lucks Lab is closely collaborating with the Sciencenter in Ithaca, NY,as part of the NSF-funded “Multi-Site Public Engagement with Science – Synthetic Biology” project. The goal of the project is to create hands-on activities for children and their families. For example, the group is working on developing a card game that can teach adults and children about the basics of synthetic biology. The cards feature images and facts about engineered microbes, such as, how microbes help humans create important medicines.

Want to have your research shared on the MCB blog? Submit your information here.