The unusual UBZ domain of Saccharomyces cerevisiae polymerase η

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.National Institute of Environmental Health Sciences (Grant ES-015818)National Institute of Environmental Health Sciences (Grant P30 ES-002109)American Cancer Society (Research Professorship

Unconventional Ubiquitin Recognition by the Ubiquitin-Binding Motif within the Y-Family DNA Polymerases ι and Rev1

Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of Y family polymerases (pol) ι and Rev1 at the stalled replication machinery is mediated by the ubiquitin-binding motifs (UBMs) of the polymerases and enhanced by PCNA monoubiquitination. We report the solution structures of the C-terminal UBM of human pol ι and its complex with ubiquitin. Distinct from other ubiquitin-binding domains, the UBM binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A, of ubiquitin abolishes UBM binding. Human pol ι contains two functional UBMs, both contributing to replication foci formation. In contrast, only the second UBM of Saccharomyces cerevisiae Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations disrupting the UBM-ubiquitin interaction also impair the accumulation of pol ι in replication foci and Rev1-mediated DNA damage tolerance in vivo.National Institute of General Medical Sciences (U.S.) (Grant GM-079376)American Cancer Society. Research ProfessorshipNational Institute of Environmental Health Sciences (Grant P30 ES-002109

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation

Structure of the ubiquitin-binding zinc finger domain of human DNA Y-polymerase η

The ubiquitin-binding zinc finger (UBZ) domain of human DNA Y-family polymerase (pol) η is important in the recruitment of the polymerase to the stalled replication machinery in translesion synthesis. Here, we report the solution structure of the pol η UBZ domain and its interaction with ubiquitin. We show that the UBZ domain adopts a classical C(2)H(2) zinc-finger structure characterized by a ββα fold. Nuclear magnetic resonance titration maps the binding interfaces between UBZ and ubiquitin to the α-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single α-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol η UBZ domain represents a novel member of the C(2)H(2) zinc finger family that interacts with ubiquitin to regulate translesion synthesis

“Filtering” by MW allows rapid MS-based proteomic identification of maltose epimerase.

<p>(A) 1-D SDS PAGE of maltose epimerase (ME) (Sigma Aldrich #M0902) resolves as a band at ∼40 kDa (boxed), the expected molecular weight for maltose epimerase. (B) Tryptic digest and MS analysis of the ∼40 kDa protein band yields a prominent doubly charged precursor ion peak at 727.84 m/z. (C) MS/MS fragmentation analysis of the 727.84 m/z peptide identified as the tryptic peptide DTPIATIGDTTGHR from the <i>L. brevis</i> protein aldose 1-epimerase. b-ion (green) and y-ion (red) series are shown. (D) Complete amino acid sequence of <i>L. brevis</i> protein aldose 1-epimerase with high confidence (>95%) MS-identified peptides colored green. Additional peptide matches are colored red.</p