DNA ligase activities during base excision repair coordination

PROJECT SUMMARY
Base excision repair (BER) is a critical mechanism for preventing the mutagenic and lethal consequences of
DNA damage generated by endogenous reactive chemical species or exposure to environmental hazards. BER
is multi-step pathway that requires a tight coordination between the repair proteins. The downstream steps of
BER pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by DNA ligase (ligase
I or IIIα). This step-to-step coordination is orchestrated by non-enzymatic scaffolding protein X-Ray Repair Cross
Complementing 1 (XRCC1) that plays a key role in assembling repair proteins. Although the roles of the individual
enzymes are largely studied, how the multi-protein BER complex coordinates while maintaining the repair
efficiency remains unclear. Though often considered an accurate process, the BER can contribute to genome
instability if normal coordination breaks down. For example, the mutations in the polβ gene that have been found
in many human cancers result in the modifications in its repair functions that impair BER efficiency. Similarly,
XRCC1 cancer-associated variants with a defective scaffolding role predispose the cell to genomic instability
and transformation. Failure in the BER pathway coordination could result in the formation of strand-break repair
intermediates that are more mutagenic or toxic than the initial DNA lesions. My research program will fill the
important gap of knowledge in the BER field by elucidating the molecular components of multi-protein BER
complex that are necessary for accurate repair and define the ramifications of defective pathway coordination
during DNA ligase I and IIIα activities. We are in a unique position to advance this scientific front based on our
strong track record and our multidisciplinary approach. In Project 1, we build off our substantial prior work using
biochemical and biophysical approach to define the molecular mechanism by which polβ, DNA ligases I and IIIα
execute the repair pathway coordination. Our studies will also elucidate cancer-associated XRCC1 and polβ
variants with altered BER functions as an important determinants of defective pathway coordination. In Project
2, using X-ray crystallography, we will elucidate the features of DNA substrate and ligase interaction that dictate
accurate versus mutagenic outcomes during final nick sealing step at atomic resolution. This project will be
extended with cryo-EM to define the structural architecture of large BER multi-protein complexes scaffolded by
XRCC1 that dictates accurate repair pathway coordination. With these 2 Projects, my laboratory will launch a
new and unique aspect of the research conducted by my group which seeks to better understand the mechanism
by which a multi-protein repair complex coordinate during BER and answer several key questions regarding how
a tight coordination is vital for maintaining the integrity of our genomic DNA, functions normally and how altering
these functions stemming from a failure in the repair pathway coordination leads to disease.

Grant Number: 7R35GM147111-05
NIH Institute/Center: NIH
Principal Investigator: MELIKE CAGLAYAN