In this study we identified PC4 as a human protein capable of suppressing the oxidative mutator phenotype of the fpg mutY
strain of E. coli
. We characterized the PC4 gene and its yeast ortholog, demonstrating that a yeast mutant devoid of its PC4 ortholog, SUB1
, is sensitive to hydrogen peroxide, exhibits a spontaneous mutator phenotype, and is hypermutable upon treatment with hydrogen peroxide. These results indicate that Sub1 is a eukaryotic peroxide resistance protein. The result demonstrating that expression of the human gene can restore peroxide resistance to the sub1Δ
mutant of yeast suggests a similar function for the human PC4 protein. These observations, combined with previous results (54
), demonstrate the utility of the bacterial screen for the identification of unique human oxidation resistance proteins.
The previously identified transcriptional coactivator function of PC4 does not require the ssDNA binding activity (56
) contained in the most highly conserved region of this family of proteins. A novel function in DNA repair for the ssDNA binding activity of PC4 is indicated by the result that the truncated form of human PC4, lacking sequences required for transcription coactivation, can function to suppress oxidative mutagenesis in bacteria and can complement the peroxide sensitivity of a yeast sub1Δ
mutant. This conclusion is further supported by the observation that the DNA binding-defective mutant forms of human PC4 are incapable of functioning as antimutators in the bacterial oxidative mutagenesis assays. Thus, we propose that PC4 functions both in transcription and in repair of oxidative DNA damage. These two functions are genetically separable; mutations in PC4's amino terminal domain primarily affect transcription coactivation, and mutations in its ssDNA binding domain primarily affect DNA repair.
Taken together, the observations that Sub1 functions in a repair pathway involving Rad2 and that PC4 directly and functionally interacts with the DNA repair protein XPG suggest a role for PC4 in the repair of oxidative damage. XPG functions in multiple DNA repair pathways in human cells. Both its enzymatic activity as a structure-specific endonuclease and a nonenzymatic function evidently involve interactions with other proteins required in NER (16
). In addition, a nonenzymatic function of XPG is required for TCR of oxidative DNA damage, evidently at an early step presumably involving recognition of RNA polymerase stalled at a lesion (34
). XPG also both stimulates BER enzymes in vitro through direct interactions (4
; B. M. Haltiwanger and P. K. Cooper, unpublished data) and stimulates removal of oxidative lesions in the cell (34
). A role for PC4 in NER is unlikely, because we have shown that deletion of its yeast homolog does not affect sensitivity to UV. Thus, the interaction of PC4 with XPG in repair of oxidative damage could conceivably affect either global BER or TCR. The stable, specific binding of XPG to double-strand DNA containing unpaired regions is functionally separate from its structure-specific endonuclease activity (25
; Sarker, Tsutakawa, and Cooper, unpublished), and an attractive possibility is that its preferential binding to the transcription-sized bubble is relevant to the TCR function of XPG. In TCR, rapid preferential repair is initiated on transcribed strands after an RNA polymerase is stalled at a DNA lesion, and it is thought that the RNA polymerase must be removed or remodeled in order for the repair enzymes to gain access to the lesion (for a review, see reference 51
). XPG is apparently required along with CSB and TFIIH for this early step in TCR (34
; S. E. Tsutakawa and P. K. Cooper, unpublished). Our finding that XPG bound to a DNA bubble substrate recruits PC4 to the complex with a resulting displacement of XPG suggests the possibility that PC4 may be involved in TCR at a step immediately following XPG.
The observed reduction of peroxide sensitivity seen when the sub1Δ rad2Δ double mutant is compared with the sub1Δ single mutant strain is consistent with the idea that Rad2 produces a potentially lethal intermediate in repair of oxidative damage that requires Sub1 for efficient further processing. Inability to release Rad2 from the partially repaired lesion (or lesion plus stalled RNA polymerase) may block access to proteins required to complete subsequent steps of repair. According to this model, the accumulation of unrepaired or inefficiently repaired intermediates leads to increased lethality in the sub1Δ mutant. Blocking production of the intermediates by eliminating Rad2 improves survival because the initial damage is not as lethal as the DNA repair intermediate. It should be noted that the effect of the rad2Δ mutation is only partial, suggesting that Sub1 may either have additional functions in DNA repair or may perform similar functions for other DNA repair enzymes.
In the context of this model, our finding that PC4 displaces XPG that is stably bound to a DNA bubble structure suggests the possibility that PC4 is required in TCR for release of XPG to allow subsequent processing either of the lesion itself or of the stalled RNA polymerase. Significantly, a requirement for an XPG release factor in NER has recently been suggested by the results of Riedl et al., who found that XPG was not released from the DNA substrate after excision of the lesion in vitro without the addition of an unknown factor present in nuclear extracts (47
). While this NER release factor is presumably not PC4, since deletion of SUB1
does not result in sensitivity to UV, it is possible that XPG similarly is not released on its own after its function in TCR but requires PC4 in order to turn over. The observation that PC4 stimulates XPG release is particularly intriguing in light of results demonstrating that PC4 can stimulate DNA synthesis via an interaction with replication protein A (44
). A possible explanation combining all these results is that PC4 may function in TCR as an intermediary between RNA polymerase removal or remodeling by XPG together with other TCR proteins, possibly clearing the initial TCR machinery from the damaged region, and subsequent steps including recruitment of repair enzymes and synthesis of the repair patch. The observation that PC4 blocks RNA polymerase elongation in vitro and the ability of TFIIH to alleviate this block (17
) raise the possibility that PC4 may also have additional functions in TCR that affect the resumption of transcription. Clearly, the close association of PC4 with other transcriptional processes and components of the transcription machinery makes a possible role for PC4 in transcription-coupled DNA repair processes particularly interesting. However, it is presently unclear if PC4 functions in XPG-stimulated BER, XPG-dependent TCR of oxidative DNA damage, or both, and further experimentation will be required to elucidate its possible roles in these processes. In this connection, it should be noted that the lack of sensitivity of the sub1
Δ mutant to UV does not rule out a requirement for PC4 in TC-NER (TCR of UV damage), since loss of TCR by deletion of RAD26 (the yeast homolog of CSB) does not render yeast sensitive to UV (53
). Thus, the postulated role for PC4 in TCR as a release factor for XPG could conceivably apply to TCR of UV and oxidative lesions.
The function of PC4 in XPG-related DNA repair processes does not readily explain the ability of this protein to function in the E. coli oxidative antimutator assay. However, since the highly sensitive E. coli antimutator assay requires only a small number of repair events per cell per generation to reveal suppression of spontaneous mutagenesis, the repair-enhancing activity of PC4 need not be very efficient. The activity we observed could be due to a general effect of PC4 binding to damaged DNA or to unpaired DNA regions produced as intermediates of repair, creating a more accessible environment for additional E. coli DNA repair factors. It is unlikely that protein-protein interactions between human PC4 protein and bacterial DNA repair proteins would be functional, and moreover, E. coli does not encode any proteins homologous to PC4. While the interaction of PC4 with XPG implicates it in an XPG-dependent DNA repair pathway in human cells, it may also function in a more general fashion to stimulate, stabilize, or assist DNA repair by other factors via a direct interaction with DNA, as it evidently can do in E. coli. Expression of human PC4 in either the wild type or the fpg mutY double mutant strain of E. coli does not result in enhanced resistance to the lethal effects of hydrogen peroxide exposure (data not shown). However, since fpg and mutY mutations have little or no effect on peroxide lethality (unpublished observations), it is unclear if this observation indicates a specificity of PC4 for repair of nonlethal lesions, such as 8-oxoG, or that the DNA repair activity required for mutation suppression when PC4 is expressed in bacteria is weak.
Sub1 mutations in yeast result in increased spontaneous and induced oxidative mutagenesis and lethality. Since expression of the PC4 protein in yeast can restore the wild-type phenotype, it suggests that the human PC4 protein may also function to prevent mutations resulting from oxidative DNA damage in human cells. Mutations that cause increased spontaneous and damage-induced mutagenesis cause an increased risk of cancer (24
). PC4 maps to chromosome 15p13, a region that frequently suffers from loss of heterozygosity in bladder and lung tumors (5
). This has been interpreted to suggest that the regulatory properties of PC4 may be important in tumor suppression, and support for this hypothesis has been presented previously (29
). However, our findings suggest an alternative, or additional, mechanism for PC4 in tumor suppression. Loss of PC4 function may also increase the rate of mutagenesis resulting from spontaneous or induced oxidative DNA damage in humans. This can increase the level of mutations leading to transformation or secondary mutations within tumors leading to tumor promotion.