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The David Lab at UC Davis utilizes numerous tools of chemical biology to explore the complex mechanistic details of DNA repair enzymes. DNA repair proteins such as MutY and NEIL, among the targets of research of the David Lab, help catalyze necessary repair of oxidative DNA damage, and are critical to maintaining genomic integrity in organisms living in the oxygen-rich environment of Earth. The David Lab is headed by Dr. Sheila S. David, who has led at the forefront of DNA repair research for over 25 years. The David Lab continues to push forward the world’s current understanding of DNA repair enzymes by leveraging our unique expertise in DNA repair enzymology while venturing into new areas and collaborating with scientists around the world.



Featured Article: 

Unique Hydrogen Bonding of Adenine with the Oxidatively Damaged Base 8-Oxoguanine Enables Specific Recognition and Repair by DNA Glycosylase MutY

Journal of the American Chemical Society

“Here we present a structure–activity relationship (SAR) study using analogues of A to probe the basis for OG:A specificity of MutY. We correlate observed in vitro MutY activity on A analogue substrates with their experimental and calculated acidities to provide mechanistic insight into the factors influencing MutY base excision efficiency.”

Click on the link or image to read more!

https://pubs.acs.org/doi/abs/10.1021/jacs.0c06767

 

Authors:
Chandrima Majumdar, Paige L. McKibbin, Allison E. Krajewski, Amelia H. Manlove, Jeehiun K. Lee*, and Sheila S. David*

J. Am. Chem. Soc. Nov. 17 2020, 142, 48, 20340–20350.



Recent Articles:

Structure, function and evolution of the Helix-hairpin-Helix DNA glycosylase superfamily: Piecing together the evolutionary puzzle of DNA base damage repair mechanisms.

The Base Excision Repair (BER) pathway is a highly conserved DNA repair system targeting chemical base modifications that arise from oxidation, deamination and alkylation reactions. BER features lesion-specific DNA glycosylases (DGs) which recognize and excise modified or inappropriate DNA bases to produce apurinic/apyrimidinic (AP) sites and coordinate AP-site hand-off to subsequent BER pathway enzymes. The DG superfamilies identified have evolved independently to cope with a wide variety of nucleobase chemical modifications. Most DG superfamilies recognize a distinct set of structurally related lesions. In contrast, the Helix-hairpin-Helix (HhH) DG superfamily has the remarkable ability to act upon structurally diverse sets of base modifications. The versatility in substrate recognition of the HhH-DG superfamily has been shaped by motif and domain acquisitions during evolution. In this paper, we review the structural features and catalytic mechanisms of the HhH-DG superfamily and draw a hypothetical reconstruction of the evolutionary path where these DGs developed diverse and unique enzymatic features.

Unique Hydrogen Bonding of Adenine with the Oxidatively Damaged Base 8-Oxoguanine Enables Specific Recognition and Repair by DNA Glycosylase MutY.

DNA repair protein MutY employs specific interactions to differentiate OG:A basepairs from canonical G:C and T:A basepairs. Prior work from our lab has focused on understanding the structural requirements of OG on lesion recognition and catalysis, and we have shown that MutY relies on the exocyclic 2-amino group of OG to identify and distinguish OG:A from other basepairs. Additionally, we’ve shown that OG binding induces conformational changes that influence A excision.

This new work uses structure-activity relationships (SARs) to identify the structural features of A that influence OG:A recognition, verification, base excision, and overall cellular repair. We correlate observed in vitro MutY activity on A analogue substrates with their experimental and calculated acidities to provide mechanistic insight into the factors influencing MutY base excision efficiency. Our results herein can be used to guide future design of MutY/MUTYH specific probes to monitor the activity, or lack thereof, of MutY/MUTYH variants. These results can also applied toward the development of MUTY/MUTYH specific inhibitors that may find utility in cancer therapeutics.

Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity

The human DNA base excision repair enzyme MUTYH (MutY homolog DNA glycosylase) excises undamaged adenine that has been misincorporated opposite the oxidatively damaged 8-oxoG, preventing transversion mutations and serving as an important defense against the deleterious effects of this damage. Mutations in the MUTYH gene predispose patients to MUTYH-associated polyposis and colorectal cancer, and MUTYH expression has been documented as a biomarker for pancreatic cancer. Measuring MUTYH activity is therefore critical for evaluating and diagnosing disease states as well as for testing this enzyme as a potential therapeutic target. However, current methods for measuring MUTYH activity rely on indirect electrophoresis and radioactivity assays, which are difficult to implement in biological and clinical settings. Herein, we synthesize and identify novel fluorescent adenine derivatives that can act as direct substrates for excision by MUTYH as well as bacterial MutY. When incorporated into synthetic DNAs, the resulting fluorescently modified adenine-release turn-on (FMART) probes report on enzymatic base excision activity in real time, both in vitro and in mammalian cells and human blood. We also employ the probes to identify several promising small-molecule modulators of MUTYH by employing FMART probes for in vitro screening.

The DNA repair enzyme MUTYH potentiates cytotoxicity of the alkylating agent MNNG by interacting with abasic sites

Inherited defects in the DNA repair gene MUTYH lead to cancer, proof that MUTYH has a critical role in preventing cancer in normal cells. In a new study from the David Lab, MUTYH is shown to have a new role that implicates it in the response to a common class of chemotherapy drugs, alkylating agents.

Cancer cells evolve resistance to chemotherapy drugs by a number of mechanisms, including upregulating DNA repair enzymes such as BRCA1, which helps cancer cells survive DNA damaging chemotherapy agents. Surprisingly, MUTYH does not help repair alkylating agent DNA damage, but instead enhance alkylating agent toxicity. This study uncovers the underlying molecular mechanism of this activity, which involves MUTYH stimulating cells to create more toxic DNA repair intermediates. By uncovering the molecular mechanism, this research suggests that MUTYH has both a role in preventing DNA mutations that cause cancer, and a separate role in helping kill cancer cells that are treated with chemotherapy drugs, thus the loss of MUTYH is a “double-whammy”. Tests to determine if cancer patients have normal versus functionally-deficient MUTYH may alter chemotherapy treatment choices if these results can be generalized to clinical practice.

Detection of OG:A Lesion Mispairs by MutY Relies on a Single His Residue and the 2-Amino Group of 8-Oxoguanine

MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYH-associated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.



Featured Photos:

Savannah completed her 3rd Year Seminar on Zoom - Congrats Savannah!

David Lab Alumni Scott Williams and family showing their BER pride.

Cindy and Liz with 2018 Nobel Laureate Frances Arnold, whose breakthrough work established the use of directed evolution to engineer enzymes.

For #NPAW2020, the David lab would like to thank Carlos for all of his hard work, mentorship, and creativity! Fun facts about Carlos, he loves glycosylases, soccer, and his family, and he has a black belt in Taekwondo! We know he will be a great PI some day!



ACS Chemical Biology LiveSlides Presentation: 

Structure–Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase

Listen in while Chandrima Majumdar explains this recent work from the David Lab, which was selected as an ACS Editor’s Choice article.



For the latest David Lab updates, check out the News section.

Click on Research for an overview of The David Lab’s research.

A thorough list of publications is available in the Publications section.



Research in the David Lab:

Liz is preparing to image her enzymatic activity assay which utilizes radiolabeled DNA.

Cindy inspects for colonies of transformed bacteria strain E. coli.

Robert pours fractions during purification of his crude compound using flash column chromatography.

Undergraduate researcher Sunbal is preparing reagents and dilutions to set up for a kinetics assay.

Kori is pipetting the necessary reagents into a well plate for her fluorescence-based enzyme activity assay.



Graduate Student Spotlight

In our second installment of David Lab Graduate Student Spotlight, you will meet Elizabeth Lotsof, a Graduate Student in Sheila David’s lab at UC Davis in the Department of Chemistry seeking to earn her Ph.D. in Chemistry. Liz focuses on DNA repair enzyme NEIL in her research. Watch now to catch her commentary on graduate school and the David Lab!


Introducing the David Lab Graduate Student Spotlight! Check out our conversation with Nicole as she discusses her journey as a Ph.D. student working in the David Lab.



Undergraduate Student Spotlight

Meet UC Davis Undergraduate Researcher Madeline Bright in our lab’s new Undergraduate Student Spotlight Video!



David Lab FYI Video

How to efficiently pour column fractions: Run a column <60 min.

Doing this will greatly increase your already-existing love of columns. And your productivity.

Get the most out of your flash column. It’s not called slow column chromatography.

Use this information at your own risk. This video is intended for graduate / professional level researchers. Be sure to follow your lab’s safety protocols.

Video by Robert Van Ostrand.

David Lab YouTube Trailer – June 2020

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A tribute to Jongchan Yeo, you are dearly missed.

Jongchan Yeo was a cherished member of the lab. He received his PhD in Chemistry in 2014 and went on to attend UC Berkeley for his post-doctoral training. He is pictured with Sheila David in his bright pink shirt that he wore while at the library for his students to find him (left) and presenting his research poster at EMGS (right). Thank you for all of your hard work.

 

His work from our lab has been published in Biochemistry.

Check it out here!



David Lab Members

The David Lab – April 2019


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Keywords: #DavidLab #TheDavidLab #UCDavis #DNA #DNARepair #Muty #Mutyh #8OG #enzymes #ModifiedOligonucleotides #ModifiedNucleosides #OrganicSynthesis #Synthesis #BaseExcisionRepair #BER #NEIL #ChemicalBiology #Chemistry #SheilaDavid #UCDavisChemistry #glycosylase #DNARepairUCDavis



 

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Contact:

Dr. Sheila S. David
ssdavid@ucdavis.edu
(530)-752-4280

Department of Chemistry
One Shields Ave.
Davis, CA 95616