p53 is a key cell cycle checkpoint factor which causes cells to undergo either cell cycle arrest or apoptosis following DNA damage. When cells are exposed to UV and X irradiation, there is an accumulation and activation of p53 (reviewed in 1
) which results in the downstream transactivation of several genes whose products assist in the maintenance of genomic integrity (4
). However, the exact mechanism of p53 activation following DNA damage is not clear. Post-translational modifications such as phosphorylation and acetylation appear to play an important role in this process (6
). Additionally, the transcriptional response of p53 requires tetramerization and this could be influenced by several factors (8
). Finally, p53 activity could be altered by interactions with other proteins. Indeed, p53 has been shown to associate in vitro
and in vivo
with a number of proteins involved in DNA metabolic processes such as repair [XPB, XPD (9
) and WRN (10
)], replication [DNA polymerase (11
) and RPA (12
)] and recombination [hRad51 (13
Although the p53 response to DNA damage is largely mediated by downstream proteins, there is increasing evidence that p53 can directly interact with DNA lesions. p53 can bind single strand and double strand breaks (14
), three-stranded DNA structures (15
), extra base bulges (17
) and several base/base mismatches in DNA (18
). Additionally, we have shown that p53 binds with extremely high affinity to Holliday junctions (16
). All of these lesions occur as a result of typical recombination, replication and repair processes as well as DNA damage caused by environmental factors. For example, Holliday junctions are natural intermediates of homologous recombination; however, high levels of sister chromatid exchange following irradiation suggests the formation of these structures due to DNA damage (19
). Replication of molecules containing thymidine dimers could result in the insertion of additional nucleotides opposite the existing lesion. Additionally, when two similar DNA molecules with a few non-identical bases undergo recombination, regions of extra base bulges and mismatches can be left behind. Replication errors can also lead to the generation of mismatches and extra base bulges in DNA. All of these lesions need to be repaired for cell cycle progression to occur. The high affinity of p53 to several of these lesions strongly points to an important role in damage recognition as well as downstream repair and recombination pathways. Thus, p53 response to lesions in DNA caused by normal metabolic processes and DNA damaging agents may be 2-fold: first, to transactive downstream genes that enable cell cycle arrest or apoptosis and, secondly, to recognize and bind these lesions to signal repair pathways.
The importance of p53 DNA binding (both sequence dependent for transactivation and sequence independent for lesion recognition) suggests that this activity is tightly regulated by a number of factors to ensure proper p53 function at the correct times during the cell cycle. p53 DNA binding activity is affected by post-translational modifications such as phosphorylation, acetylation and sumolation (reviewed in 20
). Binding by the monoclonal antibody, pAB421 (21
), as well as interaction with single strand DNA (23
) activates sequence-specific DNA binding. The identification of several cellular and viral proteins that associate with p53 suggests that protein–protein interactions play an important role in the regulation of its DNA binding activity. This is further supported by the demonstration that Ref-1 is a potent activator of p53 DNA binding activity (24
). Additionally, Jayaraman et al.
) have shown that the high mobility group (HMG) protein HMG-1 stimulates sequence-specific p53 DNA binding.
The HMG proteins are among the largest group of non-histone chromatin proteins. HMG proteins can be classified into three groups: the HMG-1/2, HMG I(Y) and HMG-14/17 families (reviewed in 26
). The exact cellular functions of these proteins are not fully understood. They are architectural elements that bind unusual structures in DNA and have low sequence specificity. The HMG-1/2 family is the most abundant of this group of proteins. They interact with DNA through two conserved DNA binding domains known as the HMG boxes (27
) and preferentially bind DNA structures that contain sharp angles such as cruciforms, four-way junctions (29
) as well as cisplatin-DNA adducts (30
). The HMG I(Y) proteins contain A-T hook domains that serve as DNA binding motifs and can recognize four-way junctions (32
). Both groups of proteins can induce bends in linear DNA templates as well as introduce supercoils in topologically constrained molecules (33
). There is growing evidence that these proteins may represent a new class of chaperone factors that can facilitate the interactions of other proteins with their respective target sequence. For example, HMG-1 induces a structural change in the target sequence of the progesterone receptor, thus facilitating protein binding (34
). Additionally, HMG I(Y) has been shown to regulate long range enhancer-dependent transcription by altering DNA topology (35
). Finally, as mentioned above, HMG-1 can enhance sequence-specific binding of p53 (25
). Thus, this group of proteins seems to enable the ‘loading’ of other proteins on their target sequences. The ability of the HMG group proteins to recognize unusual DNA structures and their influence on various protein–DNA interactions, as well as the fact that HMG-1 stimulates the sequence-specific binding of p53, makes them good candidates for factors that might affect the lesion binding properties of p53. Thus, it was critical to ask whether any of these HMG proteins influences p53 recognition of DNA damage. In this study, the effects of two HMG group proteins, HMG-1 and HMG I(Y), on p53 binding to DNA lesions were examined. Two different substrates, one containing three 3-cytosine bulges and the other resembling Holliday junctions, were used to monitor the effect of these HMG proteins on p53 DNA binding activity by gel retardation assays.
Lesions such as mismatches and extra base bulges are recognized by the Escherichia coli
mismatch repair protein, MutS, and its eukaryotic counterparts (MSH2, MSH3 and MSH6) (36
and references therein). p53 and hMSH2–hMSH6 share a number of common features. Both can recognize extra base bulges and Holliday junctions in vitro
). Both proteins can inhibit DNA recombination when they encounter DNA lesions both in vivo
and in vitro
). Additionally, these proteins play an important role in genome maintenance by interacting with other repair and replication factors. The relevance of p53 lesion recognition events within the cell was demonstrated by comparing the effect of the HMG proteins on the DNA binding activity of hMSH2–hMSH6. Our results indicate that HMG I(Y) can effectively compete with p53 and hMSH2–hMSH6 for Holliday junction binding. However, HMG I(Y) had no effect on p53 binding to 3-cytosine bulges while dissociating hMSH2–hMSH6 complexes with the same substrate. On the other hand, it was found that hMSH2–hMSH6 had a stimulatory effect on p53 binding to both Holliday junctions and 3-cytosine bulges. Thus, p53 lesion binding activity can be modulated in different ways—negative regulation is seen with HMG I(Y) and positive with hMSH2–hMSH6.