Abstract
Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.
Highlights
DNA double-strand breaks (DSBs) are caused by ionizing radiation, chemicals or replication stress and are the most cytotoxic of all DNA lesions
In order to prepare a homogeneous complex for structure determination, we performed electrophoretic mobility shift assays (EMSA) of a 165 bp nucleosome (Nuc165) with increasing molar equivalents of wild-type human Poly(ADP-ribose) Polymerase 2 (PARP2)
The complex formed with PARP2-QFRD and Nuc165 at the same molar ratio and under the same conditions tested for PARP2-WT yielded homogeneous single particles suitable for cryo-EM analysis (Fig 1B)
Summary
DNA double-strand breaks (DSBs) are caused by ionizing radiation, chemicals or replication stress and are the most cytotoxic of all DNA lesions. Their occurrence elicits a very rapid cellular response called the DNA damage response (DDR), leading to cell-cycle arrest until the break is repaired or until the cell is directed to apoptosis. Understanding the DDR is critical to optimize cancer therapies that inflict an overwhelming amount of DNA damage to tumor cells. Inhibitors of the DDR are used in combination with radiotherapy to lower the radiation dose required to efficiently kill cancer cells [1,2,3].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
