Investigating the role of HNRPUL1 in regulating the ATR-dependent DNA damage response
Lead Research Organisation:
University of Birmingham
Department Name: Cancer Sciences
Abstract
All cells have the ability to deal with potentially lethal DNA damage caused by exposure to radiation. This cellular process helps prevent mutations being inherited and requires a protein called ATR. ATR reacts to damaged DNA by stopping it from replicating. Mutations in the ATR gene, or genes that help ATR to become switched on following the generation of DNA damage can cause disorders in man with severe developmental defects (e.g. Seckel syndrome, primary microcephaly and primordial dwarfism). Therefore, understanding how ATR recognises DNA damage and stops cell growth to allow time for the damage to be repaired is essential to understand how defects in this process contribute to human disease. Our laboratory has recently identified a protein called HNRPUL1 that we have shown is required for ATR to respond properly to some forms of DNA damage. Very little is known about the role of HNRPUL1 in the cell. Therefore, our research aim is to investigate how HNRPUL1 is involved in helping ATR function. This will be carried out in a number of ways: 1). Does HNRPUL1 help the ATR protein to be activated by helping it to bind to damaged DNA? 2). Does HNRPUL1 help other proteins, required for ATR activation, bind to ATR once it has recognised DNA damage? 3). Does HNRPUL1 help cells repair DNA damage? and 4). How does HNRPUL1 help ATR to stop cell growth to allow time for the damaged DNA to be repaired? Answering these questions will increase our understanding of the cellular response to DNA damage and may reveal the involvement of the HNRPUL1 gene in the development of human disease.
Technical Summary
DNA damage recognition and repair is a fundamental process required for maintenance of the genome. DNA damage is initially recognized by a number of protein complexes that respond to different types of damage. Ataxia-telangiectasia and Rad3-related protein (ATR) responds primarily to the presence of single-stranded DNA (ssDNA) generated by the processing of DNA ends following ultraviolet (UV), ionising radiation (IR) induced damage and spontaneous damage during DNA replication. The recruitment and subsequent activation of ATR is dependent upon the interaction of its binding partner, ATR interacting protein (ATRIP), with Replication protein A (RPA) coated ssDNA. Once activated at sites of DNA damage, ATR is involved in coordinating multiple pathways required for initiating cell cycle arrest, DNA repair or apoptosis if the damage is irreparable.
We have identified the protein HNRPUL1 as being required for the efficient activation of ATR following the induction of DNA damage through its ability to enable the recruitment of RPA to DNA breaks. As a consequence, cells depleted of HNRPUL1 exhibit defective activation of cell cycle checkpoints and ionizing radiation sensitivity. Our hypothesis is that HNRPUL1 facilitates activation of the ATR-dependent DNA damage response by promoting ATR/ATRIP/RPA loading onto single-stranded DNA.
Our main objectives are:
(1) To use recombinant proteins in combination with various biotinylated DNA structures that mimic ssDNA generated during DNA end processing or replication fork stalling to assess whether HNRPUL1 facilitates the activation of ATR in vitro through its ability to either promote RPA loading on to ssDNA or ATR-ATRIP loading on to ssDNA already pre-coated with RPA.
(2) To investigate whether HNRPUL1 influences the generation of ssDNA following IR-induced damage, by monitoring BrdU incorporation into DNA under non- denaturing conditions as well as the recruitment of the nuclease complex containing Mre11 and CtIP to sites of DNA breaks.
(3) To determine the consequences of defective ATR activation in cells lacking HNRPUL1, we will assess homologous recombination repair using FACS to monitor reconstitution of a GFP expression construct in which a DNA double strand break has been introduced.
(4) To investigate whether the inability of HNRPUL1-depleted cells to activate DNA damage checkpoints is caused by defective activation and/or recruitment of known checkpoint kinases such as Chk1.
The complexity of how ATR is activated has limited our understanding of its function. An important outcome will be greater understanding of how the cell modulates ATR recruitment to sites of DNA damage.
We have identified the protein HNRPUL1 as being required for the efficient activation of ATR following the induction of DNA damage through its ability to enable the recruitment of RPA to DNA breaks. As a consequence, cells depleted of HNRPUL1 exhibit defective activation of cell cycle checkpoints and ionizing radiation sensitivity. Our hypothesis is that HNRPUL1 facilitates activation of the ATR-dependent DNA damage response by promoting ATR/ATRIP/RPA loading onto single-stranded DNA.
Our main objectives are:
(1) To use recombinant proteins in combination with various biotinylated DNA structures that mimic ssDNA generated during DNA end processing or replication fork stalling to assess whether HNRPUL1 facilitates the activation of ATR in vitro through its ability to either promote RPA loading on to ssDNA or ATR-ATRIP loading on to ssDNA already pre-coated with RPA.
(2) To investigate whether HNRPUL1 influences the generation of ssDNA following IR-induced damage, by monitoring BrdU incorporation into DNA under non- denaturing conditions as well as the recruitment of the nuclease complex containing Mre11 and CtIP to sites of DNA breaks.
(3) To determine the consequences of defective ATR activation in cells lacking HNRPUL1, we will assess homologous recombination repair using FACS to monitor reconstitution of a GFP expression construct in which a DNA double strand break has been introduced.
(4) To investigate whether the inability of HNRPUL1-depleted cells to activate DNA damage checkpoints is caused by defective activation and/or recruitment of known checkpoint kinases such as Chk1.
The complexity of how ATR is activated has limited our understanding of its function. An important outcome will be greater understanding of how the cell modulates ATR recruitment to sites of DNA damage.
