RESUMO
Eukaryotes are endowed with multiple specialized DNA polymerases, some (if not all) of which are believed to play important roles in the tolerance of base damage during DNA replication. Among these DNA polymerases, Rev1 protein (a deoxycytidyl transferase) from vertebrates interacts with several other specialized polymerases via a highly conserved C-terminal region. The present studies assessed whether these interactions are retained in more experimentally tractable model systems, including yeasts, flies, and the nematode C. elegans. We observed a physical interaction between Rev1 protein and other Y-family polymerases in the fruit fly Drosophila melanogaster. However, despite the fact that the C-terminal region of Drosophila and yeast Rev1 are conserved from vertebrates to a similar extent, such interactions were not observed in Saccharomyces cerevisiae or Schizosaccharomyces pombe. With respect to regions in specialized DNA polymerases that are required for interaction with Rev1, we find predicted disorder to be an underlying structural commonality. The results of this study suggest that special consideration should be exercised when making mechanistic extrapolations regarding translesion DNA synthesis from one eukaryotic system to another.
Assuntos
Caenorhabditis elegans/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Drosophila melanogaster/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Animais , Immunoblotting , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismoRESUMO
Recent research has revealed the presence of ubiquitin-binding domains in the Y family polymerases. The ubiquitin-binding zinc finger (UBZ) domain of human polymerase η is vital for its regulation, localization, and function. Here, we elucidate structural and functional features of the non-canonical UBZ motif of Saccharomyces cerevisiae pol η. Characterization of pol η mutants confirms the importance of the UBZ motif and implies that its function is independent of zinc binding. Intriguingly, we demonstrate that zinc does bind to and affect the structure of the purified UBZ domain, but is not required for its ubiquitin-binding activity. Our finding that this unusual zinc finger is able to interact with ubiquitin even in its apo form adds support to the model that ubiquitin binding is the primary and functionally important activity of the UBZ domain in S. cerevisiae polymerase η. Putative ubiquitin-binding domains, primarily UBZs, are identified in the majority of known pol η homologs. We discuss the implications of our observations for zinc finger structure and pol η regulation.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquitina/metabolismo , Dedos de Zinco , Animais , Sequência Conservada , DNA Polimerase Dirigida por DNA/genética , Ácido Edético/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Zinco/metabolismoRESUMO
DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol zeta, Pol kappa, Pol eta, and Pol iota.