Date of Presentation

5-5-2022 12:00 AM

College

School of Osteopathic Medicine

Poster Abstract

Uracil DNA glycosylase, or UNG2, is an enzyme that is involved in DNA repair. Its primary job is to eliminate harmful uracil bases from DNA strands. To do this, the enzyme is assisted by replication protein A (RPA). RPA helps UNG2 in the identification of uracil bases by targeting UNG2 activity near ssDNA-dsDNA junctions (1-3). The results from assays presented here agree with published findings that showed UNG2 is heavily targeted by RPA to uracil bases that are close to ssDNA-dsDNA junctions (for example, uracil located 9 bps from the junction as opposed to 33 bps) (1,2). However, these previous experiments were performed in the absence of a macromolecular crowding agent. Inert compounds such as PEG8K can be used experimentally to better represent the physiologic environment inside a cell, which is very crowded with proteins and other small molecules and differs from dilute conditions often used in enzyme assays. In the presence of a crowding agent (PEG8K), we found that RPA balances UNG2’s selectivity for uracil sites near ssDNA-dsDNA junctions that contain a 5’ ssDNA section. In other words, UNG2 becomes less targeted to uracils that are very close to the junction (i.e., U9). Interestingly, this effect was not seen when we examined RPA effects on UNG2 activity using ssDNA-dsDNA junctions substrates that contain a 3’ ssDNA section.

Keywords

Uracil-DNA Glycosidase, Replication Protein A, Uracil, Single-Stranded DNA, DNA, DNA Repair

Disciplines

Genetic Processes | Genetic Structures | Laboratory and Basic Science Research | Medical Molecular Biology | Medicine and Health Sciences

Document Type

Poster

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May 5th, 12:00 AM

UNG2 and RPA Activity on ssDNA-dsDNA Junctions

Uracil DNA glycosylase, or UNG2, is an enzyme that is involved in DNA repair. Its primary job is to eliminate harmful uracil bases from DNA strands. To do this, the enzyme is assisted by replication protein A (RPA). RPA helps UNG2 in the identification of uracil bases by targeting UNG2 activity near ssDNA-dsDNA junctions (1-3). The results from assays presented here agree with published findings that showed UNG2 is heavily targeted by RPA to uracil bases that are close to ssDNA-dsDNA junctions (for example, uracil located 9 bps from the junction as opposed to 33 bps) (1,2). However, these previous experiments were performed in the absence of a macromolecular crowding agent. Inert compounds such as PEG8K can be used experimentally to better represent the physiologic environment inside a cell, which is very crowded with proteins and other small molecules and differs from dilute conditions often used in enzyme assays. In the presence of a crowding agent (PEG8K), we found that RPA balances UNG2’s selectivity for uracil sites near ssDNA-dsDNA junctions that contain a 5’ ssDNA section. In other words, UNG2 becomes less targeted to uracils that are very close to the junction (i.e., U9). Interestingly, this effect was not seen when we examined RPA effects on UNG2 activity using ssDNA-dsDNA junctions substrates that contain a 3’ ssDNA section.

 

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