Proteins HMG-domain

Kasparkova, J. Zehnulova, J. Farrell, N. Brabec, V. DNA interstrand crosslinks of novel antitumor trinuclear platinum complex BBR. Conformation, recognition by HMG-domain proteins and nucleotide excision repair. J Biol Chem 2002, 277, 48076-48086. 

The unwinding of the helix at the site of platination is 25°. The curvature and shape of the platinated duplex are remarkably similar to those observed in DNA duplexes complexed by the HMG-domain proteins revealing that cisplatin binding alters DNA in such a manner as to facilitate HMG-domain protein recognition as mentioned in the previous paragraph. 

Dunham, S.U. and Lippard, S.J. (1997) DNA sequence context and protein composition modulate HMG-domain protein recognition of cisplatin-modified DNA. Biochemistry 36, 11428-11436. 

Huang JC, Zamble DB, Reardon JT, Lippard SJ, Sancar A (1994) HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer-drug cisplatin by human excision nuclease. Froc Natl Acad Sd USA 91 10394-10398... 

Table 3. HMG-Domain Protein-Binding Constants for Cisplatin-DNA Adducts...

The Role of HMG-Domain Proteins in Modulating the Cisplatin Sensitivity of Cells... 

If HMG-domain proteins function as a determinant of cisplatin cytotoxicity, the levels of these proteins would be expected to reflect cellular drug sensitivity. This issue is difficult to address, however, because it is not known whether only one member of this family, and if so, which one, or all HMG-domain proteins should be examined. Nevertheless, several studies have searched for a connection between the quantity of expressed HMG-domain protein and cellular response to cisplatin. No correlation was de-... 

Several models have been proposed to explain what specific role HMG-domain proteins could play in the cisplatin mechanism of action. When the ability of this family to recognize cisplatin-modified DNA was first detected, it was suggested that HMG-domain proteins might be factors that communicate the presence of the genetic damage to the repair pathways [117], but no evidence to date supports such a hypothesis. 

Fig. 5. Models for HMG involvement in the cisplatin mechanism of action. A) When a cell is exposed to a lethal dose of cisplatin, 104—105 DNA adducts are formed. If HMG-domain proteins bind with similar affinity to these lesions and to their natural binding sites, the proteins could be titrated away from their transcriptional regulatory function. B) The HMG-domain proteins could block access of the excision repair complex and...

The endogenous HMG-domain proteins in HeLa cell free extracts do not seem to affect the relative rates of repair of cisplatin-DNA adducts [54] [62], Nevertheless, the hypothesis that HMG-domain proteins can enhance cellular sensitivity to cisplatin by blocking repair of the DNA adducts is still viable. Several HMG-domain proteins are specifically expressed in the testes ([146] and references cited therein), two of which, tsHMG and hSRY, inhibit the in vitro excision of cisplatin-DNA adducts at lower protein concentrations than any of the other HMG-domain proteins tested [54] [146], Selective expression of these or other such proteins in testicular tumors would provide an explanation for the unusual cisplatin sensitivity of this tumor type and the reduced repair of cisplatin-DNA adducts observed in testicular cell lines (discussed above). 

As mentioned above, one consequence of stalled RNA polymerase II at a DNA adduct is activation of transcription-coupled repair [27], This effect may depend on the type of polymerase, however, since the removal of some types of DNA damage is slower from RNA-polymerase I transcribed ribosomal DNA than from a nuclear gene [160], The lower level of repair in the nucleolus could also reflect the influence of other transcription factors, such as the HMG-domain protein UBF, which bind to cisplatin-mod-ified DNA [145]. When HeLa cells were exposed to cisplatin at concentrations which did not seem to affect nuclear transcription, inhibition of rDNA gene expression was associated with the redistribution of UBF, along with other factors responsible for rRNA transcription [138], These observations indicate how cisplatin might exert a combination of effects. Transcription is stopped due to titration of essential factors by the platinum-DNA adducts, and the same proteins could shield the lesions from the repair activity. 

In order to understand all aspects of the action of cisplatin as an anticancer agent, further structural research on the complexes between HMG-domain proteins and platinated DNA fragments will be necessary. On the other hand, further studies of small model compounds may furnish several new details of the chemistry controlling cisplatin-DNA interactions. 

Although the platinum-amino-acid complexes do not show much promise as cytotoxic agents, these results demonstrated the utility of in vitro screening methods to survey the DNA-binding properties of platinum compounds in a combinatorial manner. Assuming that HMG-domain proteins are involved in the cisplatin mechanism of action, then screening based on the Pt-... 

A better approach would be to screen the molecular libraries on solid-phase supports [6][10][11]. Solid phase methods offer several advantages, allowing compounds to be identified by immobilization and position in a binding assay. Solid-phase screening can also be performed with the aid of robotics to increase throughput [7]. As indicated in Fig. 4, a fluorescently labeled HMG-domain protein would facilitate the search for Pt-DNA-HMG binding by solid-phase methodologies. 

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