Understanding the multivalent binding properties of PWWP2A

• Applicant: Professorin Dr. Sandra Brigitte Hake
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 409033964

Histone variants endow chromatin with unique properties and are involved in the control of all DNA-based processes. The evolutionary conserved H2A.Z variant participates in the regulation of transcription, cell cycle progression, chromosome stability and DNA damage repair. Recently, we have identified PWWP2A as a novel H2A.Z-nucleosome binding protein. PWWP2A is vertebrate-specific and contains two proline-rich stretches, an unusual internal region of no sequence homology to other proteins, a serine-rich stretch and a conserved PWWP domain. PWWP2A interacts with chromatin using a multivalent binding mode and regulates gene transcription, is crucial for metaphase-anaphase transition in human tissue culture cells and is responsible for proper cranial-facial development in Xenopus.Now, we are planning to investigate what functional role PWWP2A plays during DNA damage repair, since we have identified H2A.XS139ph (γ-H2A.X) and MDC1, which are essential in DNA damage repair, as novel PWWP2A interactors. Here, we will apply quantitative in vitro binding assays with recombinant nucleosomes and MDC1 deletions to determine how and with which domains PWWP2A binds to γ-H2A.X and MDC1. Additionally, we will utilize different in vivo approaches to understand the functional relevance of PWWP2A in recognition and/or repair of DNA damage. We will monitor live recruitment of PWWP2A to genomic site of DNA damage in a time-resolved manner. Further, we will delete PWWP2A to determine and quantitate its role in active DNA repair using several cellular reporter systems.In summary, we will apply state-of-the-art experiments to decipher PWWP2A’s binding mode to factors that mark and recognize DNA damage sites and to understand the functional relevance of the PWWP2A-MDC1-γ-H2A.X axis during DNA damage repair.

• DFG Programme: Research Grants