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. 2010 Feb 12;37(3):408-17.
doi: 10.1016/j.molcel.2009.12.038.

Unconventional ubiquitin recognition by the ubiquitin-binding motif within the Y family DNA polymerases iota and Rev1

Martha G Bomar  1 , Sanjay D'Souza, Marzena Bienko, Ivan Dikic, Graham C Walker, Pei Zhou
Affiliations

Unconventional ubiquitin recognition by the ubiquitin-binding motif within the Y family DNA polymerases iota and Rev1

Martha G Bomar et al. Mol Cell. .

Abstract

Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of Y family polymerases (pol) iota 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 iota 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 iota 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 iota in replication foci and Rev1-mediated DNA damage tolerance in vivo.

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Figures

Figure 1
Figure 1
Structure of the human pol ι UBM2. (A) Backbone traces of the NMR ensemble of 25 structures. Helices and loops are colored in red and grey, respectively. (B) Ribbon diagram. Core aromatic residues are shown in the stick model. (C) Sequence alignment of UBMs. Conserved residues are highlighted, with the signature “Leu-Pro” motif in purple, hydrophobic residues in brown and negatively charged residues in red. Pol ι UBM2 mutations evaluated for ubiquitin binding and foci formation are labeled with blue circles, and Rev1 UBM2 mutants assayed for DNA damage response are indicated by purple asterisks. The listed genes are Homo sapiens (Hs) pol ι: AF245438; Mus musculus (Mm) pol ι: AAS75834; Hs Rev1 AAI30412; Mm Rev1 AAF23323; Gallus gallus (Gg) Rev1 AAV80844; Saccharomyces cerevisiae (Sc) Rev1: NP_014991.
Figure 2
Figure 2
Structure of the human pol ι UBM2-ubiquitin complex. (A) Backbone traces of the NMR ensemble of 25 structures. Strands, helices and loops are colored in blue, red, and grey, respectively. (B) Ribbon diagram of the complex, with UBM2 in pale green and ubiquitin in orange. Side chains of ubiquitin-interacting residues are shown in the stick model. Side view (C) and front view (D) of the ubiquitin-binding interface on UBM2 depict an interaction network centered at L8, instead of I44, of ubiquitin. (E) Representative strips of intermolecular NOE crosspeaks between UBM2 and ubiquitin from the [F1] 13C-purged, [F2] 13C-separated NOESY-HSQC spectra. Only one of the two binding partners is 13C-labeled. Red asterisks indicate proton diagonal positions. Resonances of UBM2 and ubiquitin are colored in pale green and orange, respectively.
Figure 3
Figure 3
UBM-ubiquitin binding is required for pol ι to accumulate in replication foci. (A) Representative foci formation in MRC5 cells transfected with mouse FLAG-tagged WT (left), ΔUBM1 (middle) or ΔUBM2 (right) pol ι. (B) Representative foci formation in MRC5 cells transfected with human EYFP-fused WT (left) or F688A (right) pol ι. (C) Percentages of MRC5 cells with foci accumulation of human EYFP-pol ι UBM2 mutants. Error bars indicate the standard deviation of three independent measurements.
Figure 4
Figure 4
Mutations of ubiquitin-binding residues in S. cerevisiae Rev1 UBM2 impair REV1-mediated survival and mutagenesis. (A) Survival of Rev1 UBM2 point mutants after MMS treatment (0.018%) or UV irradiation (30 J/m2). (B) Reversion frequency for Rev1 UBM2 mutants after a dose of UV irradiation (15 J/m2). Error bars represent the standard deviation of three independent measurements.
Figure 5
Figure 5
A distinct mode of ubiquitin recognition by UBM. (A) Ubiquitin binding by UIM (yellow, PDB 1Q0W) and IUIM/MIU (light blue, PDB 2FID) occurs via a single helix. Ubiquitin binding by three-helix bundle domains UBA (pink, PDB 2G3Q) and GAT (light cyan, PDB 1YD8) is shown in panels (B) and (C) respectively. UBM is colored in pale green. Ubiquitin orientations are identical to that in Figure 2C or 2D.
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