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. 2006 Nov 28;103(48):18107-12.
doi: 10.1073/pnas.0608595103. Epub 2006 Nov 15.

Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen

Ildiko Unk  1 , Ildikó Hajdú, Károly Fátyol, Barnabás Szakál, András Blastyák, Vladimir Bermudez, Jerard Hurwitz, Louise Prakash, Satya Prakash, Lajos Haracska
Affiliations

Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen

Ildiko Unk et al. Proc Natl Acad Sci U S A. .

Abstract

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C(3)HC(4) RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Sequence homology of human SHPRH and yeast Rad5 proteins. (A) Schematic representation of SHPRH and Rad5 proteins. Relative positions of the conserved sequence motifs are represented by boxes. Both SHPRH and Rad5 contain SWI/SNF helicase motifs and a C3HC4 RING-finger domain, which is embedded in between the helicase motifs. The gray boxes numbered from I to VI indicate the conserved helicase motifs, whereas the conserved RING-finger domain is represented by a black box. Additionally, SHPRH also contains an H1,5 domain found in linker histone 1 and histone 5 families and a PHD domain represented by the C4HC3 motif. (B) Alignment of S. cerevisiae Rad5 and human SHPRH proteins. Residues conserved between Rad5 and SHPRH are represented by black boxes. The helicase motifs and the C3HC4 RING finger motif conserved between the two proteins are indicated. The H1,5 histone linker domain and the PHD domain, which are present only in SHPRH, are denoted by boxes. Because of the absence of any interesting features, the N terminus of the two proteins is not shown.
Fig. 2.
Fig. 2.
Physical interaction of SHPRH with Mms2–Ubc13 and Rad6–Rad18. (A) Purity of SHPRH and of Rad6–Rad18 and Mms2–Ubc13 protein complexes. Purified proteins were analyzed on a 12% denaturating polyacrylamide gel and stained with Coomassie blue. Lane 1, molecular mass standards; lane 2, 1 μg of purified SHPRH; lane 3, 1 μg of purified Rad6–Rad18 complex; lane 4, 1 μg of purified Mms2–Ubc13 complex. (B) GST pull-down of purified SHPRH with Rad6–Rad18 and Mms2–Ubc13. GST–SHPRH (3 μg) was mixed with Mms2–Ubc13 (2 μg) and Rad6–Rad18 (6 μg). After incubation, samples were bound to glutathione-Sepharose beads, followed by washings and elution of the bound proteins by glutathione. Aliquots of each sample, taken before addition to the beads (L), from the flow-through fraction (F), from the last washing fraction (W), and from the eluted proteins (E), were analyzed on an SDS/12% polyacrylamide gel stained with Coomassie blue. (C) Immunoprecipitation of human SHPRH in complex with Ubc13 from human cells. HEK293T cells were transiently transfected with plasmids expressing the epitope-tagged HA–Ubc13, alone or together with FLAG-SHPRH proteins as indicated. Thirty-six hours after transfection half of the cells were treated with 42 J/m2 UV, and 5 h later total cellular lysates were prepared. Immunoprecipitations were carried out with anti-FLAG antibodies and subsequently the precipitates were analyzed on Western blots by using anti-FLAG and anti-HA antibodies as indicated.
Fig. 3.
Fig. 3.
DNA-dependent monoubiquitylation of PCNA by Rad6–Rad18. (A) Schematic representation of DNAs used in the PCNA ubiquitylation reactions. I, 75/31-nt partial heteroduplex DNA, in which the 75-nt strand contains biotin-streptavidin bound at each end; II, single-stranded 75-nt oligomer containing biotin-streptavidin bound at each end; III, double-stranded pUC19 plasmid DNA nicked enzymatically at four positions; IV, single-stranded M13 DNA. (B) PCNA monoubiquitylation by Rad6–Rad18 in the presence or absence of DNA. The complete reaction mixture contained 50 nM PCNA, 100 nM Rad6–Rad18, 100 nM Uba1, 50 μM ubiquitin, and 10 nM RFC, along with 50 nM oligomeric DNAs (I and II) or 5 nM circular DNA (III and IV). Reactions were carried out in the absence or presence of various DNA samples (I–IV). Monoubiquitylation of PCNA was followed by Western blot using anti-PCNA antibody. (C) K164R mutant PCNA is defective in ubiquitylation. K164R mutant PCNA was subjected to ubiquitylation reaction in the presence of DNA I or III under the conditions described for wild-type PCNA.
Fig. 4.
Fig. 4.
SHPRH is a ubiquitin ligase that promotes PCNA polyubiquitylation by Mms2–Ubc13. (A) SHPRH is required for PCNA polyubiquitylation. Standard ubiquitylation reaction of PCNA (50 nM) was carried out in the presence or absence of Rad6–Rad18 (100 nM), Mms2–Ubc13 (100 nM), and SHPRH (100 nM). Combinations of these factors were omitted from the reaction as indicated on top. Monoubiquitylation and polyubiquitylation of PCNA were followed by Western blot using anti-PCNA antibody. (B) SHPRH and Mms2–Ubc13 act in a stoichiometric complex. Polyubiquitylation of PCNA (50 nM) was carried out at constant Rad6–Rad18 concentration (200 nM) but at different levels of SHPRH (5–50 nM) and Mms2–Ubc13 (20–250 nM) as indicated.
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