Biomedical subjects

Jiuya He

Publications and source records attributed to Jiuya He.

3 recordsLinked to original sources

PubMed2003

Chimeric recombinases with designed DNA sequence recognition.

Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating "custom-built" recombinases that act at chosen natural target sequences.

Amino Acid Sequence↗

PubMed2002

Interactions of protein complexes on supercoiled DNA: the mechanism of selective synapsis by Tn3 resolvase.

"Looping" interactions of distant sites on DNA molecules, mediated by DNA-binding proteins, feature in many regulated genetic processes. We used plasmids containing up to six res recombination sites for Tn3 resolvase to analyse looping interactions (synapsis) in this system. We observed that in plasmids with four or more res sites, certain pairs of sites recombine faster than others. The relative rates of recombination depend on the number, relative orientation, and arrangement of the sites. To account for the differences in rate, we propose that pairing interactions between resolvase-bound res sites are in a state of rapid flux, leading to configurations in which the maximum number of sites within each supercoiled substrate molecule are synapsed in a topologically simple arrangement. Recombination rates reflect the steady state concentrations of these synapse configurations. Our results are at variance with models for selective synapsis that rely on ordered motions within supercoiled DNA, "slithering" or "tracking", but are compatible with models that call for reversible synapsis of pairs of sites by random collision, followed by formation of an interwound productive synapse.

Binding Sites↗

PubMed2002

Synapsis of Tn3 recombination sites: unpaired sites destabilize synapses by a partner exchange mechanism.

Catalysis of site-specific recombination is preceded by the formation of a synapse comprising two DNA sites and multiple subunits of the recombinase, together with other "accessory" proteins in some cases. We investigated the stability of synapses of Tn3 resolvase-bound res recombination sites, in plasmids containing either two or three res sites. Although synapses are long-lived in plasmids with just two res sites, persisting for tens of minutes, a synapse of any two sites is relatively short-lived in plasmids with three res sites. The three alternative pairwise synapses that can be formed in three-res plasmids re-assort rapidly relative to the rate of recombination. We propose a "partner exchange" mechanism for this re-assortment, involving direct attack on a synapse by an unpaired res site. This mechanism reconciles studies on selective synapsis in multi-res substrates, which imply rapid interchange of synaptic pairings, with studies indicating that synapses of two Tn3res sites are stable.

Base Sequence↗