Genomic DNA is continuously subjected to spontaneous damage from reactive metabolites and environmental mutagens. Despite the protection provided by cellular DNA repair pathways, damaged DNA can evade detection and persist into S-phase. When this occurs, the high-fidelity polymerases δ and ε that are responsible for bulk DNA replication cannot accommodate the damage, leading replication forks to stall and posing a threat to genomic stability.

DNA translesion synthesis (TLS) is a DNA damage tolerance pathway that allows the cell to overcome replication barriers. In TLS, specialized low-fidelity polymerases utilize the damaged template to restart DNA synthesis. This process is remarkably efficient in living cells; however, these polymerases are error-prone in vitro and consequently must be carefully regulated in cells.

What mechanisms govern their recruitment? Does a lesion-specific signal recruit the “correct” polymerase? We have shown that pol η does not display a substantial preference for replicating a damaged template relative to undamaged DNA, nor for ubiquitylated PCNA relative to PCNA in vitro, casting doubt on these simple models.

Rather, the fidelity of lesion bypass might be explained by the overall architecture of the replication complex. This model is reminiscent of the concept of “replication factories”, which have been proposed to correspond microscopically observable DNA replication foci (Figure). What is the composition of these molecular complexes and how does that composition change to orchestrate the response to a particular lesion as a function of time? Can we identify specific factors associated with the response to a particular DNA lesion, associated with a particular strand (leading vs. lagging), and/or in a particular cell cycle phase (S vs. G2)?

Despite extensive biochemical investigations using traditional approaches, a conclusive answer to these questions remains elusive. To fill these gaps in our understanding, our lab is using proximity labeling techniques to elucidate a model of TLS in human cells. In these experiments, a biotin ligase is fused to a specific TLS polymerases, allowing us to monitor changes at the fork in response to DNA damage using LC/MS-MS.

Selected Publications

1. Hedglin, M., Benkovic, S.J. “Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle” (2017) Chem. Rev. 12, 7857.
2. Hedglin, M., Pandey, B., Benkovic, S.J. “Characterization of human translesion DNA synthesis across a UV-induced DNA lesion” (2016) eLife 5:e19788.
3. Hedglin M., Benkovic S.J. “Regulation of Rad6/Rad18 Activity During DNA Damage Tolerance” (2015) Annu. Rev. Biophys. 44, 207.
4. Hedglin, M., Aitha, M., Pedley, A, Benkovic, S.J. “Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen” (2019) J. Biol. Chem. 294, 13, 5157.
5. Hedglin M., Kumar R., Benkovic S.J. “Replication Clamps and Clamp Loaders.” (2013) In M. DePamphilis, S. Bell, & M. Mechali (Eds), DNA Replication (pp. 165 – 183). Long Island, NY: Cold Spring Harbor Laboratory Press.
6. Hedglin M., Perumal S.K., Hu Z., Benkovic S.J. “Stepwise Assembly of the Human Replicative Polymerase Holoenzyme.” (2013) eLife; 2:e00278.
7. Wang L, Xu X, Kumar R, Maiti B, Liu CT, Ivanov I, Lee TH, Benkovic SJ. “Probing DNA Clamps with Single-Molecule Force Spectroscopy.” (2013) Nucleic Acids Research, 41, 7804 – 7814.
8. Kumar R, Nashine VC, Mishra PP, Benkovic SJ, Lee TH. “Stepwise Loading of Yeast Clamp Revealed by Ensemble and Single-molecule Studies.” (2010) PNAS, 107, 19736 – 19741.

  Collaborators

• Kristin Eckert, Penn State Cancer Institute, Department of Pathology and Laboratory Medicine
• Ganesh Anand, The Pennsylvania State University, Faculty Director, Mass Spectrometry and Proteomics Facility

Group Members

• Dr. Philip Hanoian
• Dr. Ali Naqi Jaffary