The Lei Li Lab at IUPUI


 

Project 1. Mechanistic understanding of the SP photochemistry.

Spore-forming bacteria are responsible for a number of serious diseases in humans, including tetanus (Clostridium tetani), and anthrax (Bacillus anthracis). These endospores pose a serious threat to human health and national security, as highlighted by the 2001 anthrax attacks, where the spores of bacterium Bacillus anthracis were used. A key problem in dealing with these endospores is that they are extremely resistant to normal sterilization means such as heat, oxidizing chemicals, and UV irradiation. For instance, some Bacillus spores can be 100-fold more resistant to UV light than the corresponding vegetative cells. This highly unusual UV resistance is due to the unique spore DNA photochemistry. In spores, the genomic DNA is bound by a group of DNA binding proteins named small acid soluble proteins (SASPs), which allows the DNA to adopt an A-like conformation. This unique DNA conformation determines that upon UV irradiation, a thymine dimer, 5-thyminyl-5,6-dihydrothymine, also called spore photoproduct or SP, is generated as the exclusive DNA photoproduct.
We aim to understand the reaction mechanism for SP photo-formation. By employing deuterium labeled dinucleotide TpT, we demonstrated that SP is formed via an intramolecular H atom transfer process between two thymine residues,answering a key question in SP photoformation. By using a neutral formacetal linker instead of a phosphate between the two thymine residues, we successfully obtained the first crystal structure of SP 50 years after its discovery.These findings provide us a solid foundation to understand the SP photochemistry. We are in the process of investigating how the SASP-DNA complex in endospores controls the DNA conformation, making SP the sole photo-damage product under UV irradiation.

Project 2. Mechanistic understanding of the spore photoproduct lyase.

The SP damage in endospore genomic DNA is repaired by an enzyme spore photoproduct lyase (SPL) at the bacterial early germination phase, thus posing little threat to the spores’ survival. SPL is a member of the radical SAM superfamily, which was defined by the characteristic CXXXCXXC motif. The three C residues serve as ligands for three irons in the [4Fe-4S] cluster, with the fourth iron being coordinated by the S-adenosylmethionine (SAM) in a bi-dentate manner, with its amino and carboxylate moieties serving as the fourth and fifth ligands to the cluster. The cluster at its +1 oxidation state donates an electron to SAM to cleave its C-S bond, generating a 5′-deoxyadenosyl radical (5′-dA) to mediate the SP repair reaction.



Utilizing dinucleotide SP TpTs with the two H6 atoms selectively labeled by deuterium, we proved that it is the H6proR atom that is taken by the 5′-dA, confirming that the SPL reaction is highly stereo-selective. However, the resulting TpT radical does not take the H-atom back from 5′-dA as predicted by the previously hypothesized mechanism, but from a conserved cysteine residue, C141, in Bacillus subtilis SPL, to yield the repaired TpT. The radical is thus transfer to C141 and the resulting thiol radical must be able to mediate the rest of the enzyme catalytic cycle - namely the SAM regeneration process. We are currently investigating how the thiol radical mediates the SAM regeneration and finishes the catalytic cycle.

Project 3. Mechanistic understanding of the phenylalanine transfer RNA modification enzyme - TYW1

Human immunodeficiency virus (HIV) is a lentivirus that causes acquired immunodeficiency syndrome (AIDS). It has infected 0.6% of the world population and claimed more than 25 million lives since its discovery in 1981. In HIV infected cells, wybutosine (Y), a guanine derivative found at 37 position of eukaryotic tRNAPhe, is found missing. The lack of this guanine modification causes frameshifting to increase 400% during translation in HIV infected cells, which enables the synthesis of reverse transcriptase, the key enzyme for HIV virus replication. Further analyses revealed the lack of Y is due to the malfunction of a tRNAPhe modification enzyme TYW1, which modifies the 1-methylguanine (m1G) to imG-14, a guanine derivative with a fused tricyclic-aromatic ring. However, despite its importance to the development of AIDS, little mechanistic information is known about TYW1.
TYW1 contains a C-X3-C-X2-C motif, which is the definitive feature of the radical SAM superfamily. Supported by a NIH R21 grant, we will elucidate how TYW1 mediates the tRNAphe modification reaction as well as how the malfunction of TYW1 contributes to the development of AIDS.

 

Lab News

* Faculty from IU schools of Medicine and Nursing listen to Vice President Joe Biden open the national cancer moonshot summit, discussing goals including doubling progress in research and treatment. Lei was at the summit hosted by the IU Simon Cancer Center, with discussions including integration with the new IU Precision Health Initiative.moonshot summit

* Lei was featured in the June/July issue newsletter at IU Melvin and Bren Simon Cancer center.

*Lei was invited to give a talk "To Quantify a DNA Photolesion via LC/MS and Immunoassay” at the “Human Single Nucleotide Polymorphisms & Disease” Gordon Research Conference, June 12-17, 2016, Mount Holyoke College, South Hadley, MA.

* Lei was invited to present our SPL research at the special section honoring Nobel Lauret Professor Aziz Sancar during the American Socity for Photobiology Annual Meeting at Tempa, FL this May.

* Our studies to reveal the correlations among thymine photodimer formation, genome instability and skin cancer occurrence will be funded by the American Cancer Society via a Cancer Research Scholar grant!

* Lei has been accepted as a full member of the Indiana University Melvin and Bren Simon Cancer Center!

* Our review "Photochemistry and Photobiology of the Spore Photoproduct: A 50-Year Journey" has been chosen as the cover story by Photochem Photobiol. 

* Lei was invited to present our research at the Nucleasides, Nucleotides & Oligonucleotides GRC 2015.

* Lei has been promoted to Associate Professor with tenure effecive on July 1st, 2015.

* Our research on spore photoproduct lysase has been funded by a NSF CAREER award.

* Our paper "Reactivity of Damaged Pyrimidines: Formation of a Schiff base intermediate at the glycosidic bond of saturated dihydrouridine” has been accepted in J. Am. Chem. Soc., 2015