DNA-binding proteins as site-specific nucleases

Summary DNA‐binding proteins can be converted into site‐specific nucleases by linking them to the chemical nuclease 1,10‐phenanthroline‐copper. This can be readily accomplished by converting a minor groove‐proximal amino acid to a cysteine residue using site‐directed mutagenesis and then chemically modifying the sulphydryl group with 5‐iodoacetamido‐1,10‐ phenanthroline‐copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high‐resolution nuclear magnetic resonance and X‐ray crystal structures, and to locate binding sites of proteins within genomes

FBI-1 Can Stimulate HIV-1 Tat Activity and Is Targeted to a Novel Subnuclear Domain that Includes the Tat-P-TEFb—containing Nuclear Speckles

FBI-1 is a cellular POZ-domain–containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor–rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription