Associate Professor of Pathology and Laboratory Medicine, UNC-CH
PHD - University of Dundee, U.K.(click for Full Publication List)
Our broad long-term goal is to understand how mammalian cells maintain ordered control of DNA replication during normal passage through an unperturbed cell cycle, and when exposed to genotoxins (DNA-damaging agents). DNA synthesis is a fundamental process for normal growth and development. Accurate replication of DNA is crucial for maintenance of genomic stability. Inaccurate replication of damaged DNA may lead to mutagenesis and cancer. Moreover, many cancers display defects in regulation of DNA synthesis and it is important to understand the molecular basis for aberrant DNA replication in tumors. Since many chemotherapies specifically target cells in S-phase, a more detailed understanding of DNA replication could also allow the rational design of novel cancer therapeutics. Our lab is interested in several aspects of DNA replication control with a particular emphasis on the interplay between S-phase checkpoints and a DNA repair mechanism termed ‘Trans-Lesion Synthesis’ (TLS), as described below.
S-phase Checkpoints. 'Checkpoints' are signal transduction pathways that respond to damaged DNA and exert negative controls over cell cycle progression. The cell cycle delays triggered by checkpoints integrate DNA repair with cell cycle progression, thereby promoting genomic stability. It is widely hypothesized that cell cycle checkpoints are important tumor-suppressive mechanisms that protect against cancer-causing environmental agents that damage the genome (such as solar UV radiation and Polycyclic Aryl-Hydrocarbons or PAH).
S-phase checkpoints are activated when replication forks encounter DNA lesions such as bulky adducts. The essential protein kinases ATR and Chk1 are activated by stalled replication, and mediate an S-phase checkpoint signaling pathway that inhibits the initiation stage of DNA synthesis.
Figure 1. S-phase checkpoint regulation of Cdc45. PAH-damaged DNA (shown in red) leads to polymerase stalling, activating the checkpoint kinases ATR and Chk1. Chk1 signaling inhibits association of the initiation factor Cdc45 with origins of replication thereby resulting in inhibition of DNA synthesis.
Although we and others have shown that Chk1 is an important component of some genotoxin-induced checkpoints, relevant downstream effectors of Chk1 in the S-phase checkpoint have not been elucidated. Therefore, a major goal of our laboratory is to identify Chk1 targets that mediate the S-phase checkpoint. Our analysis of known replication proteins has identified the essential DNA replication factor Cdc45 as a target of Chk1 signaling. Studies are underway to elucidate the mechanism(s) that mediate negative regulation of Cdc45 by Chk1
Figure 2. Cdc45-induced chromatin decondensation is inhibited by BPDE (a DNA-damaging PAH).
Trans-Lesion Synthesis (TLS) DNA Polymerases. TLS polymerases are specialized enzymes that synthesize DNA with low fidelity on undamaged DNA templates, yet replicate damaged DNA with relatively high accuracy. Following exposure to genotoxins TLS polymerases are recruited to sites of DNA damage thereby preserving replication fork movement.
Figure 3. Nuclear distribution of the TLS polymerase Pol-kappa (green foci) and sites of replication fork stalling (red foci) in a PAH-treated cell.
Different TLS polymerase perform replicative bypass of specific DNA lesions. For example, a TLS enzyme termed 'Polymerase eta' (Pol-eta) uniquely performs bypass of UV-induced cyclobutane pyrimidine dimers. Failure to recruit appropriate TLS polymerases to sites of DNA damage can result in incomplete replication and replication fork collapse or error-prone mutagenic replication by a ‘wrong’ polymerase. Defects in Pol-eta can cause the human disease xeroderma pigmentosum (XP) which is characterized by extreme sensitivity to sunlight and propensity to UV-induced skin cancers.
We are interested in mechanisms of TLS polymerase regulation. An E3 ubiquitin ligase termed Rad18 is important for guiding TLS polymerases to sites of stalled replication and ubiquitinating PCNA (a replication fork component and DNA polymerase processivity factor), thereby facilitating engagement of TLS polymerases with replication forks.
Figure 4. PCNA mono-ubiquitination regulates 'polymerase switching' at stalled replication forks. (1) A DNA lesion (shown in red) causes stalling of the replicative DNA Polymerase Pol-gamma. The E3 ligase Rad18 guides Pol-eta (aTLS DNA polymerase) to stalled replication forks. (2) Rad18 monoubiquitinates PCNA at stalled replication forks. (3) Pol-eta engages mono-ubiquitinated PCNA and performs replicative bypass of damaged DNA, preserving replication fork movement.
Mechanisms that regulate Rad18 and recruit TLS polymerases in a lesion-specific manner are incompletely understood. We are studying protein kinases that phosphorylate Rad18 and coordinate its activity with cell cycle and checkpoint signaling. Additionally, we are performing screens to identify new Rad18 substrates.
Our studies of Cdc45 and Rad18 regulation (described above) represent individual aspects of broader ongoing work to elucidate the complex series of events that regulate DNA replication when cells acquire DNA damage, and also during an unperturbed S-phase.
- Guo, N., Faller, D. V., and Vaziri, C. Carcinogen-Induced S-phase is Chk1-Mediated and Caffeine-Sensitive. Cell Gr. Diff. 13, 77-86 (2002)
- Weiss, R., Leder, P., and Vaziri, C. A Critical Role for Mouse Hus1 in a S-phase DNA Damage Cell Cycle Checkpoint. Mol. Cell Biol. 23, 791-803 (2003)
- Vaziri, C., Saxena, S., Jeon, Y., Lee, C., Lee, C., Murata, K., Machida, Y., Wagle, N., Hwang, D. S., and Dutta, A. A p53-Dependent Pathway Prevents Re-replication. Mol. Cell 11, 997-1008 (2003)
- Bi. X, Slater, D. M., Ohmori, H., and Vaziri, C. DNA Polymerase Kappa Is Specifically Required For Recovery From The Benzo[a]Pyrene-Di-hydrodiol Epoxide (BPDE)-Induced S-phase Checkpoint. J. Biol Chem. 280, 22343-22355 (2005)
- Bi, X., Barkley, L. R., Tateishi, S., Yamaizumi, M., Ohmori, H., and Vaziri, C. Rad18 Regulates DNA Polymerase Kappa and is Required Recovery From The BPDE-Induced S-phase Checkpoint. Mol. Cell Biol. 26, 3527-35240 (2006)
- Liu, P., Barkley, R. Bi, X., Slater, D. M., Alexandrow, M., Nashuer, H. P., and Vaziri, C. The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/CDK2-independent mechanism. J. Biol. Chem - In 281, 30631-30644 (2006)
- Barkley, L. R., Ohmori, H. and Vaziri, C. (2007) Integrating S-phase Checkpoint Signaling with Trans-Lesion Synthesis of Bulky DNA Adducts. Cell Biochem Biophys. 47(3):392-408.
- Tomida, J., Masuda, Y., Hiroaki, H., Ishikawa, T., Song, I., Tsurimoto, T., Tateishi, S., Shiomi, T., Kamei, Y., Kim, J., Kamiya, K., Vaziri, C., Ohmori, H., and Todo, T. (2008) DNA Damage Induced Ubiquitylation of RFC2 Subunit of RFC Complex. J. Biol. Chem. 283, 9071-9079
- Ohashi, E., Hanafusa, T., Kamei, K., Song, I., Vaziri, C. and Ohmori, H (2009) Identification of Novel REV1-interacting Motifs Necessary for Y-family Polymerase Function. Genes To Cells 14, 101-11.
- Liu, P., Slater, D.M., Lenburg, M., Nevis, K., Cook, J.G., and Vaziri, C. (2009) Replication Licensing Promotes Cyclin D1 Expression and G1 Progression in Untransformed Human Cells. Cell Cycle 8, 125-36
- Barkley, Song, I, and Vaziri, C. (2009) Reduced Expression of GINS Complex Members Induces Replication Stress and Activates an ATM/Chk2-Mediated Checkpoint. Cell Cycle 8, 1577-88