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Functional Characterisation of APLF; A Novel Human Protein Involved in the Cellular Response to Chromosomal DNA Strand Breaks

Reference	BB/F013930/1
Principal Investigator / Supervisor	Professor Keith Caldecott
Co-Investigators / Co-Supervisors
Institution	University of Sussex
Department	Genome and Damage Stability Centre
Funding type	Research
Value (£)	404,340
Status	Completed
Type	Research Grant
Start date	01/07/2008
End date	31/08/2011
Duration	38 months

Abstract

DNA strand breaks are a major threat to cell survival and genetic integrity. Single-strand breaks (SSBs) arise from a variety of sources including oxidative stress and are the commonest DNA lesions arising in cells (tens of thousands per cell per day). If not repaired, SSBs can block transcription and can be converted into potentially clastogenic or lethal DNA double-strand breaks (DSBs) during DNA replication. Recently, we identified an uncharacterized open reading frame (C2orf13) that encodes a novel human protein that we denoted Aprataxin and PNK-Like Factor (APLF), based on its sequence similarity to the single-strand break repair (SSBR) and double-strand break repair (DSBR) proteins Aprataxin and PNK. Our preliminary experiments have demonstrated that, similar to Aprataxin and PNK, APLF is recruited into the SSBR and DSBR machinery through CK2-mediated phosphorylation-dependent interactions with the scaffold proteins XRCC1 and XRCC4, respectively. Moreover, these experiments revealed that APLF rapidly accumulates at sites of DNA strand breakage in human cells and that depletion of APLF significantly reduces rates of both SSBR and DSBR following oxidative stress or ionizing radiation. These data identify APLF as an exciting new component of the cellular response to DNA damage. In the current proposal we will examine the phenotype of cells lacking and/or depleted of APLF and characterise the function of this exciting new protein at the biochemical, cellular, and physiological level.

Summary

The DNA in our cells is damaged more than 10,000 times per cell per day. If not repaired properly, this damage can result in genetic mutations and/or cell death. Consequently, cells have evolved a number of sophisticated biochemical mechanisms by which damaged DNA is rapidly detected and repaired. Recently, my group identified a completely novel human DNA repair protein that we denoted Aprataxin and PNK-Like Factor, or APLF. We have discovered that APLF is important for ensuring that breaks in one or both strands of DNA are repaired as fast as possible. Here, we plan to characterise in detail the importance of APLF for genetic integrity and genetic stability in living cells and in animals. In addition, we will identify the biochemical function of APLF and integrate this role into our model for how DNA strand breaks are repaired in human cells.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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