The single-strand DNA binding activity of human PC4 prevents mutagenesis and killing by oxidative DNA damage

Abstract

Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1Delta mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

FIG. 1.

Mutator phenotype of E. coli fpg mutY and its suppression by bacterial and human DNA repair genes. Upper panels show the phenotype of (A) wild-type E. coli cc104 carrying the GC→TA transversion-specific allele of lacZ and (B) its isogenic fpg mutY double mutant derivative. The antimutator activity resulting from (C) expression of bacterial fpg protein (E. coli fpg mutY/pfpg), (D) expression of the human 8-oxoG DNA glycosylase OGG1 (E. coli fpg mutY/phOGG1), (E) expression of the human MutY ortholog hMYH (E. coli fpg mutY/phMYH), and (F) expression of the truncated form of PC4 isolated in this study (E. coli fpg mutY/pSE380-PC4).

FIG. 2.

Structure of PC4 and its derivatives. The upper panel shows the domains of wild-type PC4. The protein region designated aa 22 to 87 is the minimal coactivator clone described by Kaiser et al. (28), and it is shown for comparison. The initial PC4 clone is the form of PC4 initially isolated in our screen. The white amino-terminal box indicates the in-frame vector sequence fused to the 40 to 127 aa region of PC4. The PC4-CTD expressed in yeast was constructed by adding an ATG codon 5′ to sequences encoding PC4 aa residues 40 through 127. The S. cerevisiae SUB1 gene is also shown, the boxes containing the dotted lines (not to scale) depict the heterologous 39-aa amino-terminal and 187-aa carboxyl-terminal domains of unknown function.

FIG. 3.

ssDNA binding activity of PC4 is required for mutation suppression in E. coli fpg mutY. Histidine-tagged forms of PC4 and its ssDNA binding-defective mutants W89A and β2β3 are expressed from the l-arabinose-inducible araBAD promoter present on the pBAD24 vector. (A) Upper panels show the mutator activity in the absence of l-arabinose, lower panels show the mutator activity of wild-type and mutant forms of PC4 after induction by l-arabinose. (B) Left section shows the Western blotting using antihistidine antibody to determine levels of wild-type (WT) and mutant protein expression; the right section shows the same gel stained with Coomassie brilliant blue.

FIG. 4.

Peroxide sensitivity of the yeast sub1Δ mutant strain, its suppression by yeast SUB1, and truncated PC4 gene expression. (A) ⧫, S. cerevisiae wild-type (wt) yeast carrying the vector p416-GPD; □, S. cerevisiae sub1Δ mutant carrying the vector p416-GPD; ▴, wild type carrying the full-length SUB1 gene; ○, S. cerevisiae sub1Δ mutant carrying the full-length SUB1 gene expression plasmid. (B) ⧫, S. cerevisiae wild-type yeast carrying the vector pMV611; □, S. cerevisiae sub1Δ mutant carrying the vector pMV611; ▴, wild type carrying the truncated PC4 gene expression plasmid; ○, S. cerevisiae sub1Δ mutant carrying the truncated PC4 gene expression plasmid.

FIG. 5.

MMS and UV survival in the wild type (wt) and sub1Δ mutant of S. cerevisiae. (A) MMS survival of wild-type yeast (○) and the sub1Δ mutant (•). Data shown represent the averages of three experiments. Error bars indicate standard errors of the mean and are shown when they extend beyond the symbol. (B) UV spot test. Overnight cultures, diluted to inoculate each spot with approximately 1,000 cells of the wild type (upper row) and the sub1Δ mutant (lower row), were placed on YPD agar plates and exposed to increasing doses of UV.

FIG. 6.

The yeast sub1Δ mutation results in elevated mutagenesis. Spontaneous and induced mutagenesis in the wild type (wt) and the sub1Δ mutant of S. cerevisiae. The insert expands the view of the mutagenesis in the absence of exogenous peroxide addition.

FIG. 7.

Partial suppression of sub1Δ hydrogen peroxide sensitivity by rad2Δ. ○, wild type (wt); ▴, sub1Δ mutant strain; ▾, rad2Δ mutant strain; ⧫, sub1Δ rad2Δ double mutant strain. Representative data are shown.

FIG. 8.

Interaction of PC4 and XPG. Slot blot and far-Western tests were performed as described in Materials and Methods. Full-length hNTH1 and E. coli EndoIII were used as the positive and negative controls, respectively. The left portions of the panels show Ponceau S-stained images of the membrane, and right portions show the results of incubation of membranes with ³²P-labeled XPG-HMK. The position of the protein in the membrane is indicated.

FIG. 9.

(A) XPG protein enhances PC4 binding to DNA bubble substrates. Portions (2.5 nM) of the 10-nucleotide (nt) bubble DNA substrate were incubated with 41 nM purified XPG (lanes 2 to 7) or without XPG (lanes 9 to 13) as described in Materials and Methods. Reaction mixtures were supplemented with 44 (lanes 3 and 9), 88 (lanes 4 and 10), 176 (lanes 5 and 11), 352 (lanes 6 and 12), and 704 nM (lanes 7 and 13) human PC4 protein. Samples were loaded onto a 4.5% native gel, and electrophoresis was conducted at 150 V for 2 h at 4°C. The gel was dried and exposed on a phosphorimager screen. No protein was added in lanes 1 and 8. (B) Quantitative representation of the effect of XPG on PC4 binding. (C) Displacement of XPG by PC4 does not depend on the order of addition of the proteins. In all experiments, 44 nM XPG and 352 nM PC4 protein and the same DNA bubble substrates shown in Fig. 8 were used. Lanes 2 and 3, XPG was first incubated with the DNA bubble substrate (lane 2) and PC4 protein was then added (lane 3); lane 4, PC4 protein was first incubated with the DNA bubble substrate followed by XPG addition; lane 5, XPG and PC4 were mixed first and then added to the DNA bubble substrate; lane 6, PC4 alone was incubated with the substrate. Samples were run in a 4.5% native gel, and electrophoresis was conducted at 150 V for 2 h in the cold. The gel was dried and exposed on a phosphorimager screen.