SUMMARY 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 ζ, Pol κ, Pol η, and Pol ι.

The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.

SummaryEukaryotes 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 S. cerevisiae or S. 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.

CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosphohydrolase family. The active site for native CD39 is located on the outer surface of the cellular plasma membrane; however, it is not yet known at what stage this enzyme becomes active along the secretory pathway to the plasma membrane. In this study, sucrose density fractionations performed on CD39-transfected COS-7 cell membranes suggest that CD39 activity resides primarily in the plasma membrane. Furthermore, we have created recombinant, soluble versions of CD39, one that is secreted and others that are retained in the endoplasmic reticulum, to demonstrate that CD39 is not active until it reaches the plasma membrane both in yeast and COS-7 cells. Moreover, the secreted active soluble CD39 in COS-7 cells is found to receive a higher degree of N-glycan addition than the inactive form retained intracellularly. When COS-7 cells were treated with tunicamycin to prevent N-glycosylation, soluble CD39 was not detected in the extracellular medium and remained inactive intracellularly. Surface biotinylation analysis also revealed that surface-expressed wild type CD39 receives a higher degree of N-glycosylation than intracellular forms and that inhibition of N-glycosylation prevents its plasma membrane localization. In addition, both intact and digitonin-permeablized COS-7 cells transfected with CD39 possess similar ecto-ATPase activities, further supporting the conclusion that only surface-expressed CD39 is enzymatically active. All of these data suggest that intracellular CD39 is inactive and that only a fully glycosylated CD39 has apyrase activity and is localized at the cell surface.

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 S. 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.