Anti-S phase agents like doxorubicin, gemcitabine, cytarabine and hydroxyurea are in extensive use for the treatment of cancers and of hematological diseases such as essential thrombocytopenia, polycythemia vera and sickle cell anemia. All these agents are believed to cause replication fork stalling leading to activation of checkpoint pathways that inhibit cell proliferation. Yet, not much is known about how these inhibitors inhibit the origins of replication, of the sites/DNA lesions responsible for checkpoint activation and of the effect of these lesions on chromosome structure or gene expression. In addition, many of these agents are known to affect gene expression by unknown mechanisms. For example, hydroxyurea has been used in sickle cell anemia because it increases the expression of fetal hemoglobin and decreases the expression of cell-surface adhesion molecules. We have recently published genomic approaches to study time of replication, origins of replication and effects of anti-S phase agents on the replication origins in human cells We have discovered that two anti-S phase agents used for therapy lead to the firing of clustered neo-origins, leading to a high density of stalled replication forks at defined sites in the genome. In this proposal we plan to determine whether this is a general feature of other anti-S phase agents, whether the sites where these clustered neo-origins occur are the same in different cell lines or whether the sites change when cells are exposed to these agents in all parts of the S phase. We will investigate whether the high density of stalled replication forks at clustered neo-origins make these sites of the genome susceptible to double-strand breaks (DSB) and chromosomal rearrangements. We will examine whether the disturbed state of the chromatin due to the high density of clustered neo-origins leads to suppression of gene expression. Finally, we will examine which, where and in what order checkpoint activators and DNA repair proteins are recruited to the stalled replication forks created by anti-S phase agents.
Anti-S phase agents like doxorubicin, hydroxyurea, gemcitabine and cytarabine are in extensive use for the treatment of cancers and hematological diseases such as essential thrombocytopenia, polycythemia vera and sickle cell anemia. Yet, very little is known about which parts of the genome they affect, the nature of the lesions on DNA, or how they interfere with the replication apparatus. As a result, it has been difficult to investigate their effects on chromosomal fragility, changes in gene expression or mechanisms by which they engage the checkpoint and repair apparatus. Based on novel techniques developed in the lab, we are now in a position to address these critical questions. The results are expected to open new avenues of research in the use of anti-S phase agents and suggest methods to improve the therapy of cancers and hematological diseases by anti-S phase agents.
|Shibata, Etsuko; Dar, Ashraf; Dutta, Anindya (2014) CRL4Cdt2 E3 ubiquitin ligase and proliferating cell nuclear antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase. J Biol Chem 289:23056-64|
|Dar, Ashraf; Wu, David; Lee, Nicholas et al. (2014) 14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation. Mol Cell Biol 34:4049-61|
|Im, Jun-Sub; Keaton, Mignon; Lee, Kyung Yong et al. (2014) ATR checkpoint kinase and CRL1?TRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks. Genes Dev 28:875-87|