1. Introduction
Protein acetylation was originally recognized as an important post-translational modification of histones during transcription and DNA repair [
1]. Recently, however, the arena of acetylation has been extended to include non-histone proteins, particularly those involved in the process of DNA double strand break (DSB) repair [
2–
5]. In fact, it has been recently demonstrated that acetylation regulates the key DNA damage response kinases ATM and DNA-PKcs [
2,
4], as well as a plethora of DNA repair factors including NBS1, Ku70, and p53 [
3,
6–
9]. These evidences tend to support a pivotal role for acetylation in the process of DNA damage response and repair—ostensibly through facilitating the recognition and signaling of DNA lesions, as well as orchestrating protein interactions to recruit activities needed in the process of the repair. Specifically, acetylation is critical in the activation of DNA damage response pathways [
2,
4]. In spite of these advances, precise functional roles of acetylation of the most non-histone DNA repair proteins are still elusive. Recent research suggests that this covalent protein post-translational modification could also confer new functional properties, and thus modified proteins can carry out distinct roles. Indeed, it has been well documented that Ku70 and p53 acetylation are involved in promoting apoptosis [
6,
8,
10]. While p53 and Ku70 interaction is acetylation-independent, p53 acetylation facilitates the dissociation of BAX from Ku70 and therefore enhances apoptosis [
7]. Due to these observations, it is presently believed that non-histone acetylation is widely spread and modulates a multitude of protein functions [
2].
This widespread pattern of protein acetylation is conceivably maintained through the action of many lysine acetyltransferases. To date, the known acetyltransferases can be classified into three families (
i.e., Gcn5/PCAF, p300/CBP, and MYST) on the basis of their amino acid sequence similarity [
5]. Over the past several years, an increasing number of lysine acetyltransferases have been implicated in the process of DNA damage response and repair mainly through modification of non-histone proteins. For example, p300/CBP and PCAF are involved in mediating DNA damage response [
6]. Likewise, the MYST acetyltransferases Tip60 (
i.e., 60 kDa Tat-interactive protein) and hMof (
i.e., males absent on the first) participate directly in DNA damage repair through controlling the functions of ATM, DNA-PKcs, p53, and c-Abl [
11–
14].
Although there is ample evidence underscoring the necessity of acetylation in DSB repair, the extent of protein acetylation in DNA damage repair is still unclear. In this study, we demonstrate that the human MutS homologue hMSH4 undergoes DNA damage-induced acetylation. Despite the fact that hMSH4 is a member of the MutS protein family [
15], to date there is no evidence for its participation in conventional mismatch repair MMR [
16]. Cumulated evidence, however, has suggested a role for hMSH4 in meiotic recombinational DSB repair [
16–
19]. In
C. elegans, silencing of BRCA1 orthologue on a MSH4-deficient background leads to chromosome fragmentation during meiosis [
20], indicating a potential synergistic effect between hMSH4 and BRCA1 on DSB processing. It is known that hMSH4 interacts with an array of protein factors—which currently include hMSH5, hMLH1, hMLH3, hRad51, DMC1, GPS2, VBP1, and eIF3f—associated with diverse cellular functions [
16,
21–
29]. This hMSH4 protein interaction profile is not only compatible with a role of hMSH4 in DSB repair, but also supports the idea that hMSH4 may exert multiple functions through interacting with different protein partners. In the present study, we have investigated DNA damage-induced hMSH4 acetylation and deacetylation, and have identified new hMSH4-interacting proteins that are responsible for these post-translational modifications and their roles in NHEJ-mediated DSB repair.
3. Discussion
It has been recently recognized that lysine residues of non-histone proteins—involved in many different biological processes including DNA damage recognition and repair—are frequently acetylated in a reversible fashion. In fact, most protein acetylation is controlled by both histone acetyltransferases (HATs) and HDACs; therefore, the levels of acetylation can be quickly adjusted to tailor protein functions in response to cellular requirements. Our current study demonstrates that hMSH4 becomes acetylated in response to IR-induced DNA damage. This DNA damage-triggered hMSH4 acetylation is mediated by hMof—one of the well-known DNA damage response acetyltransferases [
35]. The tissue expression profiles of hMSH4 and the MYST family acetyltransferases,
i.e. hTip60 and hMof, are very similar [
36], which supports the idea that the interplay of these proteins could exist in a variety of cell types. In addition, our study has also demonstrated that hMSH4 can be deacetylated by HDAC3. Collectively, our data indicate that hMSH4 acetylation is dynamically regulated by hMof and HDAC3. Consistent with observations implicating hMSH4 in the HR process, both hMof and HDAC3 are known to play important roles in the process of DSB repair [
11,
34]. This supports a scenario in which both acetylation and deacetylation attribute to the function of hMSH4 in DSB repair.
The results of our present study also suggest that hMof antagonizes the suppressive effect of hMSH4 on the mutagenic NHEJ-mediated DSB repair. In conjunction with the known protein interaction profile of hMSH4 with HR proteins [
16], hMSH4 acetylation could likely serve as a mechanism to regulate protein-protein interaction during DNA damage recognition and repair. Given the constitutively low levels of hMSH4 expression in human cells [
15,
25], acetylation might temporally change hMSH4 protein stability and/or conformation, presumably through the competition with lysine polyubiquitination—a modification known to mediate hMSH4 degradation [
37]. Furthermore, the timing of hMSH4 acetylation in response to DNA damage may be also pertinent to the role of hMSH4 in the repair process.
Several studies have linked hMSH4 to disease conditions in humans. A recently study reported that
hMSH4 expression in the breast cancer cell line MCF-7 was down-regulated due to DNA hypermethylation [
38]. The
hMSH4 non-synonymous SNP G
289→A (
i.e., encoding hMSH4
Ala97Thr) has been associated with an increased risk for breast cancer [
39], while
hMSH4 G
1243→A (
i.e., encoding hMSH4
Glu415Lys) has been identified as an important marker for blood malignancy [
40]. Studies in
C. elegans have previously shown that the orthologues of hMSH4 and BRCA1 acted synergistically in the maintenance of chromosome stability [
20]. In addition, loss of chromosomal region 1p31-32, harboring
hMSH4 and several other genes, in myeloma patients is significantly associated with shorter survival [
41]. These observations have underscored the possibility that hMSH4 is important for the maintenance of chromosome stability even though it is normally expressed at a very low level.
Since the hMSH4 and hMof interaction in human cells occurs only after the induction of DNA damage, the basal level of hMSH4 acetylation is likely to be maintained by acetyltransferases through transient interactions. It is plausible that, in addition to hMof, hGCN5 may potentially contribute, at least to certain extent, to the basal hMSH4 acetylation. Although the role of induced hMSH4 acetylation in DNA damage response still remains to be defined, the results of our current study have also raised several other interesting possibilities. First and foremost, this DNA damage-induced hMSH4 acetylation might play a role in the regulation of protein-protein interactions. Thus, it would be critical to determine whether hMSH4 acetylation poses any effects on its interaction with hMSH5—an altered hMSH4-hMSH5 interaction can potentially exert a significant impact on the interplay of hMSH5 with c-Abl in DNA damage response and repair [
30,
42,
43]. This is also pertinent to the catalytic outputs of c-Abl in regulating the balance between DSB repair and the activation of cell death response [
42,
44,
45]. Finally, the nuclear functions of hMSH4 and its interacting partner hMSH5 are likely harnessed by mechanisms governing nuclear-cytoplasmic protein trafficking [
46]. Therefore, it would be interesting to know whether hMSH4 acetylation may have any effect on nuclear-cytoplasmic protein redistribution. Answers to these questions will certainly lead to new avenues for future studies of the biological functions of hMSH4 in DSB damage response and repair processes.