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Novel ubiquitinated PCNA probes for understanding DNA damage tolerance
Date
2021
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Publisher
University of Delaware
Abstract
Chapter 1: Ubiquitin is a 76-amino acid protein and is highly conserved in eukaryotes. Ubiquitination is one of the most complex protein post-translational modification. Ubiquitin is conjugated to the substrate protein through a cascade of enzymatic reactions. Ubiquitination plays an important role in regulating proteasomal degradation, chromatin remodeling, cell cycle, immune response, protein trafficking and DNA damage response. In addition to monoubiquitination, ubiquitin can form polyubiquitin chains through one of its seven lysine residues. For example, the most well-known function of ubiquitin in regulating protein degradation is through the Lys48 polyubiquitination. Both mono- and K63-linked poly-ubiquitinations are formed on a nuclear protein proliferating cell nuclear antigen (PCNA), which serve as signals following the stalling of replication fork during DNA replication. PCNA and its mono- or poly ubiquitination are involved in different DNA damage tolerance pathways (error-prone versus error-free). We are interested in characterizing the reader proteins for mono- or poly-ubiquitinated PCNA in regulating DNA damage tolerance. ☐ The Y-family DNA polymerase η (Polη) is one of the critical readers for mono-ubiquitinated PCNA at the stalled replication fork. Polη synthesizes pass damaged DNA nucleotide in eukaryotes through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of PCNA, which signals the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. Through photo-crosslinking the probes with full-length Polη, specific crosslinking site(s) were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη’s catalytic core and C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance. ☐ Chapter 2: It is important to obtain high-resolution structures of the eukaryotic Y-family DNA polymerases such as Polη in complex with PCNA or Ub-PCNA. We combined photo-crosslinking and chemical ubiquitination to generate photo-activatable Ub-PCNA probe for structural studies of the yeast Polη/Ub-PCNA complex. I generated the (pBpa)Ub and the (pBpa)Ub-PCNA probes for complex formation with Polη. Different chromatography methods including ion exchange, nickel or cobalt affinity, heparin and size exclusion were assessed for purification of the Polη/Ub-PCNA complex. The development of (pBpa)Ub-PCNA probe, generation and purification of Polη/Ub-PCNA complex open the door for obtaining a high resolution structure of the complex through the state-of-the-art cryo-EM methodology. ☐ Chapter 3: Maintenance of genome stability 1 (Mgs1) is another important Ub-PCNA reader that preferentially recognizes polyUb-PCNA. Mgs1 is a DNA-dependent AAA(+) ATPase and is conserved from prokaryotes to eukaryotes. Mgs1 is an oligomeric protein with yet unknown number of subunits. Mgs1 has been shown to promote genome stability when the replication fork encounters DNA damage during replication. However, the exact mechanism of Mgs1’s biochemical and cellular functions are unclear. Using our photo-activatable PCNA or Ub-PCNA probes, I unveiled the interaction between Mgs1 and PCNA on both the front and back sides of the PCNA ring. The interaction sites between PCNA and Mgs1 were identified using mass spectrometry. Preliminary electron microscopy study of Mgs1 was attempted. Together the studies provided useful information regarding the structure and function of Mgs1 in DNA damage tolerance.
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Keywords
Probe, Ubiquitin, Cell cycle, Genome stability