Nonrandom integration of retroviral DNA in vitro: effect of CpG methylation.

AUTOR(ES)

Kitamura, Y

RESUMO

We have developed a PCR-based system that allows us to assess the relative frequency of use of specific bases as targets for the avian leukosis virus in vitro integration system. Using this system, we tested the effect of 5-methylation of cytosine in runs of CpG on the distribution of integration target sites. We found that the distribution of preferred integration sites was not uniform along the target DNA; rather, there was a distinct and reproducible pattern of frequently used sites. This pattern was independent of orientation of the integrated DNA, and of overall structure and sequence of the target and fragment amplified. Methylation did not inhibit integration into CpG dinucleotides; on the contrary, this modification created highly preferred targets within runs of alternating CpG. Finally, similar but not identical specificity was observed by using preintegration complexes in infected extracts or purified integrase and DNA as enzyme and substrate. Thus, most of the specificity observed is conferred by interaction of integrase and targets, although it may be modified by other viral and/or cellular components.

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