Cyrus Vaziri, Ph. D.

Adjunct Associate Professor
Department of Pathology and Laboratory Medicine



B.S. University of London, U. K. (1989) Ph.D. University of Dundee, U.K. (1992)

Vaziri Lab Research Interests

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 in response 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. Many cancers display defects in regulation of DNA synthesis and it is important to understand the molecular basis for aberrant DNA replication in tumors. Moreover, since many chemotherapies specifically target cells in S-phase, a more detailed understanding of DNA replication could allow the rational design of novel cancer therapeutics. Our lab focuses on three main aspects of DNA replication control: I. The S-phase checkpoint, II. Trans-Lesion Synthesis (TLS) and III. Re-replication, as described below.

I. Molecular Basis of the Bulky Adduct-Induced S-phase Checkpoint.

Our lab’s interest in S-phase regulation stems from studies we initiated several years ago to investigate biological effects of the ubiquitous environmental pollutant and potent human carcinogen benzo[a]pyrene or B[a]P (Fig. 1) . B[a]P from combustion products (including tobacco smoke, car exhaust) is oxidized within cells generating benzo[a]pyrene dihydrodiol epoxide (BPDE), a metabolite that binds DNA covalently to form ‘bulky adducts’. This form of DNA damage can lead to mutagenesis and cancer. Therefore, B[a]P and related adduct-forming genotoxins pose a serious threat to human health. ‘Checkpoints’ are signal transduction pathways that respond to damaged DNA by exerting negative controls over cell cycle progression. The cell cycle delays triggered by checkpoints integrate DNA repair with cell cycle progression, thereby maintaining genomic stability. There is good evidence that cell cycle checkpoints are important tumor-suppressive mechanisms that protect against cancer. S-phase checkpoints are activated when replication forks encounter DNA lesions such as B[a]P adducts. We have now elucidated many components of the S-phase checkpoint that inhibits DNA synthesis in response to B[a]P adducts. We have shown that the essential protein kinases ATR and Chk1 are components of a B[a]P-induced S-phase checkpoint signaling pathway that inhibits the initiation step of DNA synthesis (Fig. 2).

Although we and others have shown that Chk1 is an important component of some genotoxin-induced checkpoints [1-3], all relevant downstream effectors of Chk1 in the S-phase checkpoint have not been identified. Therefore, a major goal of our laboratory is to identify Chk1 targets that mediate the B[a]P-induced S-phase checkpoint. Our analysis of known replication proteins has identified the essential DNA replication factor Cdc45 as a target of Chk1 signaling [2]. Studies are underway to elucidate the mechanism(s) that mediate negative regulation of Cdc45 by Chk1. Our studies of the S-phase checkpoint have led to a broader interest in mechanisms by which Cdc45 is regulated both during a normal cell cycle and in the DNA damage response. Identification of links between Chk1, Cdc45 and S-phase control will provide new insight into control of normal DNA replication as well as mechanisms of tumor suppression. Moreover, understanding mechanisms of Chk1 action might identify new therapeutic targets in proliferative disorders. As described in II below, we have identified the ubiquitin ligase Rad18 as another novel Chk1 target that is involved in tolerance of B[a]P-induced DNA damage.

II. Trans-Lesion Synthesis (TLS) DNA Polymerases and the S-phase Checkpoint.

One of the most important recent findings in the field of chemical carcinogenesis was the discovery of specialized DNA polymerases termed the ‘Trans-Lesion Synthesis’ (TLS) enzymes. TLS polymerases synthesize DNA with low fidelity on undamaged DNA templates, yet replicate damaged DNA with relatively high accuracy. Defects in a TLS enzyme termed ‘Polymerase eta’ (Pol eta) can cause the human disease Xeroderma Pigmentosum (XP). XP is characterized by extreme sensitivity to sunlight and propensity to UV-induced skin cancers. We have found that a specific TLS polymerase, DNA Polymerase Kappa (Pol Kappa) is recruited to sites of B[a]P-induced replication fork stalling [4, 5], as shown in Fig. 3.

Fig. 3 Nuclear distribution of Polk (green foci) and sites of DNA replication (red foci) in a B[a]P-treated cell .

Pol kappa-mediated bypass of B[a]P adducts enables attenuation of checkpoint signaling and resumption of normal S-phase progression (Fig. 4). It is likely that Pol kappa plays a role in preventing cancers induced by environmental exposure to the ubiquitous carcinogen B[a]P.

Fig. 4 Role of Polk in the B[a]P-induced S-phase checkpoint. I. B[a]P-adducted DNA (shown in red) causes stalling of the replicative DNA polymerase (Pol) and activates the S-phase checkpoint. II. A ‘polymerase switch’ replaces the replicative DNA polymerase for the TLS polymerase Polk. III. Polk-mediated bypass of the B[a]P lesion allows recovery of the stalled replication fork and attenuates S-phase checkpoint signaling.

We demonstrated recently that the E3 ubiquitin ligase Rad18 mono-ubiquitinates Proliferating Cellular Nuclear Antigen (PCNA) in response to B[a]P adducts [4]. PCNA is a replication fork component that tethers polymerases to the DNA. PCNA mono-ubiquitination constitutes the ‘polymerase switch’ that disengages replicative polymerases and recruits Pol kappa to sites of DNA damage. However, the mechanism by which DNA damage triggers Rad18-dependent PCNA ubiquitination is not clear. We have found that ATR/Chk1 checkpoint signaling is required for efficient PCNA mono-ubiquitination and subsequent recruitment of Pol kappa to stalled replication forks [4]. These results demonstrate novel mechanisms for integrating checkpoint signaling with TLS. We hypothesize that Rad18 is a direct or indirect target of Chk1 and that Rad18 regulation by Chk1 is a key step in TLS. Experiments are underway to determine the mechanisms by which Chk1 signaling regulates Rad18. Our studies of Cdc45 and Rad18 regulation (described above) represent individual aspects of broader studies to elucidate the complex series of events that regulate DNA replication when cells acquire DNA damage. The studies described in I and II above deal with consequences of DNA damage from extrinsic sources. In contrast, work described in section III (below) is focused on the effects of inherent problems with DNA replication, specifically the detrimental consequences of DNA re-replication.

III. A Novel DNA ‘Re-replication Checkpoint’.

To maintain genomic stability, it is important that each chromosome undergoes only a single round of DNA replication per cell cycle. Potentially, over-replication of the genome could lead to gene amplification, one of the hallmarks of cancer cells. In studies that stemmed from our lab’s interest in the DNA damage-induced S-phase checkpoint, we discovered a novel checkpoint mechanism that restricts DNA synthesis to ‘once-per-cell cycle’ [6]. We noticed that aberrant expression of the DNA replication factor Cdt1 elicited a dramatic re-replication phenotype in cancer cells but not primary untransformed cell lines. In primary cell lines, Cdt1 induced a growth arrest that was mediated by the p53 tumor suppressor protein. We were able to confer Cdt1-dependent re-replication in normal cells when we perturbed p53 function [6]. These studies demonstrated that p53 plays a key role in preventing over-replication of the genome in response to aberrant replication factor activity. The activation of p53 in response to over-expressed Cdt1 may result from DNA damage-mimetic ‘onion skins’ generated during re-replication (Fig. 5).

Fig. 5 Consequences of re-replication in normal and cancer cells.

More recently, we have found that another important human tumor suppressor, the Retinoblastoma (RB) protein may also prevent re-replication in cells with excess Cdt1 activity. These results suggest that p53 and Rb, two known tumor suppressors that are inactivated in most human cancers, exert their tumor-suppressive activities in part by preventing DNA re-replication. We are currently dissecting the precise mechanisms by which p53 and Rb prevent DNA re-replication and genetic instability. In summary, we are working on several inter-related projects that will elucidate molecular details of the fundamental biological process of DNA replication. DNA replication is a highly coordinated step in normal growth and development, and its regulation is perturbed in proliferative disorders such as cancers. In fact, tumor cells are often sensitive to agents that perturb DNA replication. Therefore, in addition to providing information regarding mechanisms of normal cell growth, tumor suppression and carcinogenesis, our studies could help to understand modes of action of existing chemotherapies and might lead to the identification of new therapeutic targets.

Current Lab Members

Tovah Day (Ph. D. Student) E-mail: Tovah is investigating the regulation of Cdc45 via post translational modifications.
Ihnyoung Song (Ph.D. Student) Email: Ihnyoung is testing the role of the Fanconi Anemia (FA) proteins in DNA replication and TLS.

Selected References

1. Guo, N., D.V. Faller, and C. Vaziri, Carcinogen-induced S-phase arrest is Chk1 mediated and caffeine sensitive. Cell Growth Differ, 2002. 13(2): p. 77-86.

2. Liu, P., et al., The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25a/Cdk2-independent mechanism. J Biol Chem, 2006.

3. Weiss, R.S., P. Leder, and C. Vaziri, Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint. Mol Cell Biol, 2003. 23(3): p. 791-803.

4. Bi, X., et al., Rad18 Regulates DNA Polymerase {kappa} and Is Required for Recovery from S-Phase Checkpoint-Mediated Arrest. Mol Cell Biol, 2006. 26(9): p. 3527-40.

5. Bi, X., et al., DNA polymerase kappa is specifically required for recovery from the benzo[a]pyrene-dihydrodiol epoxide (BPDE)-induced S-phase checkpoint. J Biol Chem, 2005. 280(23): p. 22343-55.

6. Vaziri, C., et al., A p53-dependent checkpoint pathway prevents rereplication. Mol Cell, 2003. 11(4): p. 997-1008.