Novel functions of the oncofetal HMGA2 protein in genome stability

• Main Author: Yu, Haojie
• Other Authors: Peter Droge
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/55737
• Institution: Nanyang Technological University

The non-histone chromatin factor HMGA2 (High Mobility Group AT-hook 2), which contains three AT-hook motifs as independent DNA binding domains, is normally expressed in ES cells and during embryonic/fetal development. In this study, I investigated its function in protecting the stalled replication forks. I found that RPA32 (Replication Protein A 32 kDa subunit) co-localises with HMGA2 at on-going or Hydroxyurea (HU)-induced stalled replication forks. The similar results were observed that IdU pulse-labeled replication sites co-localise with HMGA2 signals in the absence and in the presence of HU. Moreover, HMGA2 protects against fork-associated DSBs (Double Strand Breaks) formation as well as degradation of nascent DNA strands after Hydroxyurea (HU)-induced fork stalling. This, in turn, facilitates fork restart, significantly suppresses chromosomal instabilities, and enhances cell viability. Strikingly, in a heterologous cell system Saccharomyces cerevisiae (S. cerevisiae), human HMGA2 alone partially complements the fork-stabilizing function of ATR/Mec1 by reducing the occurrence of pathological fork structures and their subsequent endonucleolytic cleavage into DSBs. Moreover, wild type HMGA2 rather than the DNA-binding motif AT-hook mutants is sufficient to fully complement the Escherichia coli RecA protein in its replication fork-stabilizing role. By employing a hexapeptide wrwycr which specifically binds to and blocks proper processing of YSs (Y-Structures) and HJs (Holliday Junction-Structures) in vitro and in vivo, HMGA2 was found to suppress peptide-induced cleavage at branched DNA structures in vivo. The faithful and complete replication of genome is essential to maintain genome integrity and prevent the accumulation of cancer-promoting mutations. The results of my study thus uncovered an important novel replication fork chaperone activity in mammalian cells which employs direct physical stabilization of branched DNA by forming a scaffold via multiple DNA binding domains in cis. This chaperone seemingly evolved to preserve ES cell genome integrity, but is hijacked by tumor (stem) cells in the adult organism to guard their genomes also against DNA-damaging agents widely used in the clinic.