SUBSTRATE-LENGTH-DEPENDENT ACTIVITIES OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE IN-VITRO - DIFFERENTIAL DNA-BINDING AFFINITIES ASSOCIATED WITH DIFFERENT LENGTHS OF SUBSTRATES

被引:23
作者
LEE, SP [1 ]
CENSULLO, ML [1 ]
KIM, HG [1 ]
HAN, MK [1 ]
机构
[1] GEORGETOWN UNIV,MED CTR,DEPT BIOCHEM & MOLEC BIOL,WASHINGTON,DC 20007
关键词
D O I
10.1021/bi00032a015
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human immunodeficiency virus type 1 integrase (HIV-IN) is an enzyme essential for the integration of viral DNA into the host chromosome, a process that is an attractive target for drug development. In vitro assays have been developed to study both components of the integration process, the 3'-processing and strand transfer reactions, However, major discrepancies between results obtained from in vivo and in vitro events raise concerns as to the biological relevance of activities observed in vitro. These discrepancies include the size of the substrate and the nature of the divalent cation used, In this study, we characterized activities of HIV-IN with oligonucleotide substrates varying in length. Our previous studies indicate that the preferred cation in vitro for 3'-processing is altered from Mn2+ to Mg2+ by increasing the length of the oligonucleotide substrate. This study demonstrates that HIV-IN efficiently catalyzes Mg2+-dependent 3'-processing while repressing the strand transfer reaction. Substrate competition studies indicate that longer substrates preferentially bind Co the viral DNA binding site of the integrase, whereas the shorter substrate has much less specificity. In addition, the shorter substrate requires a higher concentration of Mg2+, indicating that there is an alteration in the metal binding affinity associated with the varying substrates. Our results show that substrate-length-dependent differential activities are due to differences in the divalent metal binding and DNA binding affinities associated with the different substrates. These results suggest that the structure of the viral DNA is an important factor in differentiating the donor and target substrates. Characterization of DNA substrates for in vitro assays may resolve some of the in vitro and in vivo discrepancies and provide further understanding of structure/function relationships between IN and DNA.
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页码:10215 / 10223
页数:9
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