Binding interaction of HMGB4 with cisplatin-modified DNA.
Proteins in the HMG family are important transcription factors. They recognize cisplatin-damaged DNA lesions with a structure-specific preference and account for more than 70% of all proteins that interact with the cisplatin 1,2-intrastrand d(GpG) cross-link. HMGB4, a new member of the mammalian HMGB protein family expressed preferentially in the testis, was generated recombinantly, and its interactions with cisplatin-modified DNA were investigated in vitro. The binding affinities of the two individual DNA-binding domains of HMGB4 to DNA carrying a cisplatin 1,2-intrastrand d(GpG) cross-link are weaker than those of the DNA-binding domains of HMGB1. Full-length HMGB4, however, has a 28-fold stronger binding affinity (K(d) = 4.35 nM) for the platinated adduct compared to that of HMGB1 (K(d) = 120 nM), presumably because the former lacks a C-terminal acidic tail. The residue Phe37 plays a critical role in stabilizing the binding complex of HMGB4 with the cisplatin-modified DNA, as it does for HMGB1. Hydroxyl radical footprinting analysis of the HMGB4/platinated DNA complex reveals a footprinting pattern very different from that of HMGB1, however, revealing very little binding asymmetry with respect to the platinated lesion. An in vitro repair assay revealed that HMGB4, at 1 μM, interferes with repair of cisplatin 1,2-intrastrand cross-link damage by >90% compared to control, whereas HMGB1 at the same concentration inhibits repair by 45%. This repair inhibition capability is highly dependent on both the binding affinity and the size of the proteins. The putative role of HMGB4 in the mechanism of action of cisplatin, and especially its potential relevance to the hypersensitivity of testicular germ cell tumors to cisplatin, are discussed.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.
Semi
S
Park
Stephen J
SJ
Lippard
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D002945:Cisplatin / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D024002:HMGB Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding
D018736:DNA Adducts; D024002:HMGB Proteins; C539347:HMGB4 protein, mouse; D004247:DNA; D002945:Cisplatin