Analysis of Xenopus Np95 during the cell cycle and in DNA damage responses

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DNA is constantly subjected to damage caused by a wide variety of different factors. If this damage is left unrepaired then mutations can arise. If allowed to persist these mutations can lead to a loss of genome integrity. In order to protect the genome, cells have evolved mechanisms to prevent DNA damage or problems encountered during DNA replication from generating mutations. These mechanisms collectively known as DNA damage responses, detect damaged DNA or stalled DNA replication and can initiate a series of responses such as DNA repair, cell cycle arrest (allows time for repair), or if damage is too extensive, programmed cell death (apoptosis). Failure of DNA damage response pathways have been implicated in the development of cancer and in several human genetic diseases associated with a predisposition to cancer as well as developmental and neurological abnormalities. Understanding how these pathways operate and impinge on other cellular processes is therefore vital if human diseases such as cancer and many human developmental abnormalities are to be understood. Using cell-free extracts of amphibian eggs that recreate cell cycle events in the test tube we will study the role of a nuclear protein known as Np95. Np95 is upregulated in many types of cancer cell and cells lacking Np95 protein are highly sensitive to DNA damaging agents and drugs that inhibit DNA replication. These observations suggests that Np95 is also necessary for efficient DNA damage responses. We will use the Xenopus cell-free extract system to determine how and when Np95 function is required during the cell cycle and whether removing Np95 from egg extracts affects the cellular response to DNA damage.