Biomedical subjects

Y V Kil

Publications and source records attributed to Y V Kil.

4 recordsLinked to original sources

PubMed2000

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).

Adenosine Triphosphate↗

PubMed1998

Tn5/IS50 target recognition.

This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5/IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5/IS50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn5/IS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.

Amino Acid Sequence↗

PubMed1994

DNA length, bending, and twisting constraints on IS50 transposition.

Transposition is a multistep process in which a transposable element DNA sequence moves from its original genetic location to a new site. Early steps in this process include the formation of a transposition complex in which the end sequences of the transposable element are brought together in a structurally precise fashion through the action of the element-encoded transposase protein and the cleavage of the element free from the adjoining DNA. If transposition complex formation must precede DNA cleavage (or nicking), then changing the length of the donor DNA between closely spaced ends should have dramatic effects on the frequency of the transposition. This question has been examined by studying the effects of altering donor DNA length on IS50 transposition. Donor DNA < or = 64 bp severely impaired transposition. Donor DNA > or = 200 bp demonstrated high transposition frequencies with only modest length dependencies. Constructs with donor DNA lengths between 66 and 174 bp demonstrated a dramatic periodic effect on transposition (periodicity approximately 10.5 bp).

Base Sequence↗

PubMed1992

Riboflavin operon of Bacillus subtilis: unusual symmetric arrangement of the regulatory region.

Seventeen cis-dominant mutations leading to riboflavin overproduction in Bacillus subtilis were localized to the region between nucleotides +37 and +159 relative to the transcription initiation site of the riboflavin operon. This region displays an unusual structure for regulatory sequences. The main part of it represents clusters of A/T and G/C-rich sequences that symmetrically blank a short inverted repeat.

Bacillus subtilis↗