Tracking of Progressing Human DNA Polymerase Holoenzymes Reveals Distributions of DNA Lesion Bypass Activities
Open Access
- Author:
- Cardina, Joseph
- Area of Honors:
- Chemistry
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Mark Hedglin, Thesis Supervisor
Lauren Dell Zarzar, Thesis Honors Advisor - Keywords:
- DNA Damage Tolerance
Human pol delta
DNA lesions
Translesion DNA synthesis
Lesion Bypass - Abstract:
- In lagging strand DNA synthesis, DNA polymerase δ (pol δ) holoenzymes composed of pol δ and the proliferating cell nuclear antigen (PCNA) sliding clamp perform high fidelity DNA synthesis with little error. The pol δ holoenzyme occasionally encounters structural deformities in the native DNA sequence called DNA lesions. Upon encountering these lesions, it was previously believed that pol δ would stall on the DNA template at some point before the lesion launching a DNA damage tolerance (DDT) pathway to replicate the lesion and remaining DNA before allowing pol δ to resume its typical high fidelity DNA synthesis. Recent literature within the past 20 years or so has challenged this perspective by showing human pol δ can replicate various DNA lesions. These studies have questioned the role that DDT plays in lagging strand DNA synthesis upon encounter of a DNA lesion, thus providing a need for further analysis. As a result, we aimed to quantitatively characterize the encounters of pol δ with several biologically relevant DNA lesions at physiological conditions. Our results show that pol δ supports dNTP incorporation while inserting across from, extending from, and elongating past several different DNA lesions, and the exact extent of these values is dependent on the exonuclease activity of pol δ and the identity of the lesion itself. We also found that of the pol δ that dissociates upon encountering the lesion, the dissociation events are unevenly distributed around the lesion with different distributions depending on the lesion. Our findings show that lagging strand DNA synthesis is more complex than previously thought while advancing our understanding of the role of DDT in lagging strand DNA synthesis.