Steven Berberich (Committee Member), Paula Bubulya (Committee Member), Madhavi Kadakia (Committee Member), Michael Leffak (Advisor), Courtney Sulentic (Committee Member)
Doctor of Philosophy (PhD)
This work investigates two questions regarding DNA replication. The first aim examines the interactions of DUE-B with replication proteins and the second explores the cellular effects of replicating a polypurine polypyrimidine sequence in human cells.
DUE-B siRNA decreases the chromatin binding of essential replication proteins Cdc45, PCNA and RPA. DUE-B also co-immunoprecipitates with Cdc45 and TopBP1. In vitro kinase assays suggest that the checkpoint protein ATR may phosphorylate DUE-B. These experiments lend further evidence that DUE-B plays an important role in the initiation of eukaryotic DNA replication.
To investigate the effects of replicating a polypurine-polypyrimidine sequence prone to secondary structure (PKD1), our laboratory constructed four cell lines. In TTR and TTF cells, the PKD1 sequence is inserted in opposite orientations in place of the triplex-prone region of the c-myc replicator (which is stably integrated into the same genomic locus in all four cell lines). In DTR and DTF cells, the PKD1 sequence is inserted in opposite orientations in place of the DNA Unwinding Element of the c-myc replicator. The probability of secondary structure formation by the PKD1 sequence varies based on the orientation due to changes in direction of replication.
In these cells, the orientation of the insert affects the cells' growth rate, overall health, dependence on a functional checkpoint, and ability to replicate DNA after synchronization with aphidicolin or mimosine. A nearby origin is not required for these effects.
When the PKD1 insert was removed from TTR cell lines, the normal phenotype was partially restored. These cells showed an intermediate response to aphidicolin and mimosine synchronization and to checkpoint inhibition.
These conclusions argue for the efficiency of replication origins and suggest that the differences between the two cellular phenotypes are caused by the direction of replication fork movement through the polypurine-polypyrimidine sequence. Importantly, these data indicate that a single genomic lesion may have the ability to alter cell growth and recovery from DNA damage.
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