Melike Caglayan, Ph.D., an assistant professor in the department of biochemistry and molecular biology in the University of Florida College of Medicine, has been awarded a $1.8 million grant from the National Institute of General Medical Sciences to study the repair mechanism of DNA.
This grant will fund research into a DNA repair process called base excision repair, or BER. This repair process is a critical mechanism for preventing mutagenic and lethal consequences of DNA damage.
“These studies will have important ramifications on current designs to develop DNA repair capacity assays as a strategy to assess cancer susceptibility or therapeutic efficacy,” Caglayan said. “Thus, defining the molecular determinants that dictate BER accuracy, particularly in the context of pathway coordination, is critical to fully understand disease mechanisms and to define how defects in the system contribute to disease risk.”
The BER pathway, a suppressor of somatic mutations, is a multistep process that requires tight coordination between several repair proteins. The downstream steps of the BER pathway involve gap filling by DNA polymerase beta, or polβ, and subsequent nick sealing by a DNA ligase. This step-to-step coordination is orchestrated by a non-enzymatic scaffolding protein called X-ray repair cross complementing 1, or XRCC1.
Although the roles of these individual enzymes are largely studied and the BER process is considered to be accurate, it is unknown how these proteins coordinate among themselves. Understanding this coordination is important because coordination failures cause further damage to DNA.
For example, mutations in the polβ gene found in many human cancers result from modifications in its BER repair function. Similarly, some XRCC1 cancer-associated variants with a defective scaffolding role predispose the cell to genomic instability.
The newly funded research at the Caglayan lab will seek to understand both the molecular components of the multi-protein BER complex and define the ramifications of defective pathway coordination during DNA ligase activities. This project will build on previous research and take a multidisciplinary approach involving biochemistry, biophysics, X-ray crystallography and cryogenic electron microscopy, or cryo-EM.
Using X-ray crystallography, Caglayan and her team will study the ligase interactions with nick DNA repair intermediates containing mismatch, ribonucleotide or damaged ends that dictate the final nick sealing step at atomic resolution. This will provide a greater understanding of the differences between accurate and mutagenic repairs. The team will also use a cryo-EM approach to better understand the mechanism by which a multi-protein repair complex scaffolded by XRCC1 coordinates during BER.
This research could answer several key questions about how proteins coordinate during BER and why this is vital for maintaining the integrity of genomic DNA. It could also lead to new insights that explain how failures in the BER pathway lead to disease and cancers.