Biomedical subjects

Michael W Kaiser

Publications and source records attributed to Michael W Kaiser.

3 recordsLinked to original sources

PubMed2004

A preliminary solubility screen used to improve crystallization trials: crystallization and preliminary X-ray structure determination of Aeropyrum pernix flap endonuclease-1.

Crystallization of protein and protein complexes is a multi-parametric problem that involves the investigation of a vast number of physical and chemical conditions. The buffers, salts and additives used to prepare the protein will be present in every crystallization condition. It is imperative that these conditions be defined prior to crystal screening since they will have a ubiquitous involvement in the crystal-growth experiments. This study involves the crystallization and preliminary analysis of the flap endonuclease-1 (FEN-1) DNA-repair enzyme from the crenarchaeal organism Aeropyrum pernix (Ape). Ape FEN-1 protein in a standard chromatography buffer had only a modest solubility and minimal success in crystallization trials. Using an ion/pH solubility screen, it was possible to dramatically increase the maximum solubility of the protein. The solubility-optimized protein produced large diffraction-quality crystals under multiple conditions in which the non-optimized protein produced only precipitate. Only minor adjustments of the conditions were required to produce single diffraction-quality crystals. The native Ape FEN-1 crystals diffract to 1.4 A resolution and belong to space group P6(1), with unit-cell parameters a = b = 92.8, c = 80.9 A, alpha = beta = 90, gamma = 120 degrees.

Aeropyrum↗

PubMed2004

Position-dependent function of a B block promoter element implies a specialized chromatin structure on the S.cerevisiae U6 RNA gene, SNR6.

The Saccharomyces cerevisiae U6 RNA gene, SNR6, is transcribed by RNA polymerase III (Pol III), but lacks the intragenic B block promoter element found in most other Pol III transcription units. Rather, the SNR6 B block element is located 120 bp downstream of the terminator. In contrast, the Schizosaccharomyces pombe U6 RNA gene has an intragenic B block sequence in a short intron. We show that the S.pombe U6 intron, when inserted into SNR6, can functionally replace the downstream B block in vitro but not in vivo. The in vivo expression defect is caused by at least three different effects of the insertion: (i) the S.pombe intron is inefficiently spliced in S.cerevisiae due to the short distance between the 5' splice site and branchpoint; (ii) the S.pombe B block sequence is suboptimal for S.cerevisiae; and (iii) a B block does not function well within the context of the SNR6 intron, especially when the gene is present at its normal chromosomal locus rather than on a plasmid. This last observation suggests that the chromatin structure of the SNR6 locus favors utilization of a downstream B block element. We also provide evidence that splicing of U6 RNA reduces its activity, presumably due to alterations in U6 RNA structure, localization and/or assembly into the spliceosome.

Alleles↗

PubMed2003

Modeling of flap endonuclease interactions with DNA substrate.

Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex and were used as distance restraints in MD simulations. The final structure suggests that the substrate's 5' flap interacts with the enzyme's helical arch and that the helix-hairpin-helix motif interacts with the template strand in the downstream duplex eight base-pairs from the cleavage site. This model suggests specific interactions between the 3' end of the upstream oligonucleotide and the enzyme. The proposed structure presents the first detailed description of substrate recognition by structure-specific 5' nucleases.

Base Sequence↗