RESUMO
The influence of chromatin structure on cisplatin DNA damage was investigated in intact human cells. The epsilon-globin gene promoter was utilised as the target DNA sequence and the terminal transferase-dependent PCR technique was employed to examine adduct formation at base pair resolution. It was found that cisplatin preferentially damaged at runs of consecutive guanine bases in intact cells. By comparing the relative intensity of adduct formation in intact cells and in purified genomic DNA, it was possible to assess the influence of chromatin proteins on the extent of cisplatin DNA damage. Enhanced damage in intact cells was found at the CACC site where a member of the Sp1 family of proteins is thought to bind. It is postulated that protein binding at this site bends the DNA double-helix so that enhanced cisplatin binding occurs. The altered DNA binding of cisplatin in the presence of chromatin proteins could be important in the properties of cisplatin as an anti-tumour drug.
Assuntos
Antineoplásicos/farmacologia , Cromatina/química , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular , Cisplatino/metabolismo , DNA/metabolismo , Adutos de DNA/química , Pegada de DNA , Desoxirribonuclease I , Eritrócitos , Globinas/genética , Guanina/química , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Fator de Transcrição Sp1/metabolismoRESUMO
An automated sequencer was used to determine the sequence specificity of DNA damage caused by hedamycin in the plasmid pUC19 using a linear amplification/Taq DNA polymerase method. Previously, manual DNA sequencers have been in widespread use to investigate the sequence specificity of a DNA damaging agent. Manual DNA sequencers are restricted in the length of DNA sequence that can be read at base pair resolution for densitometry. An automated sequencer can greatly expand on the length of analysable DNA sequence. An additional important capability of the automated sequencer, is the ability to quantitate the intensity of damage at each base pair site. Thus we have used the automated sequencer to elucidate the sequence specificity of DNA damage for 300 bp. We have carried out an extended analysis of the sequence specificity of hedamycin DNA damage and found that the sequence 5'-cGt-3', tGt and cGg are preferentially damaged. The sequence specificity of cisplatin was also investigated.