Biomedical subjects

Stuart J Haring

Publications and source records attributed to Stuart J Haring.

4 recordsLinked to original sources

PubMed2006

Functional assays for replication protein A (RPA).

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein. RPA is conserved in all eukaryotes and is essential for DNA replication, DNA repair, and recombination. RPA also plays a role in coordinating DNA metabolism and the cellular response to DNA damage. Assays have been established for many of these reactions. This chapter provides an overview of the methods used for analyzing RPA-DNA interactions, RPA-protein interactions, and functional activities of RPA. Methods are also discussed for visualizing RPA in the cell and analyzing the effects of RPA function on cell cycle progression in mammalian cells.

Chromatography, Affinity↗

PubMed2004

A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae.

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.

Amino Acid Motifs↗

PubMed2004

The signal from the initiation of meiotic recombination to the first division of meiosis.

Two of the unique events that occur in meiosis are high levels of genetic recombination and the reductional division. Our previous work demonstrated that the REC102, REC104, REC114, and RAD50 genes, required to initiate meiotic recombination in Saccharomyces cerevisiae, are needed for the proper timing of the first meiotic (MI) division. If these genes are absent, the MI division actually begins at an earlier time. This paper demonstrates that the meiotic recombination genes MER2/REC107, SPO11, and MRE2 and the synaptonemal complex genes HOP1 and RED1 are also required for the normal delay of the MI division. A rec103/ski8 mutant starts the MI division at the same time as in wild-type cells. Our data indicate no obvious correlation between the timing of premeiotic S phase and the timing of the first division in Rec- mutants. Cells with rec102 or rec104 mutations form MI spindles before wild-type cells, suggesting that the initiation signal acts prior to spindle formation. Neither RAD9 nor RAD24 is needed to transduce the signal, which delays the first division. The timing of the MI division in RAD24 mutants indicates that the pachytene checkpoint is not active in Rec+ cells and suggests that the coordination between recombination and the MI division in wild-type cells may occur primarily due to the initiation signal. Finally, at least one of the targets of the recombination initiation signal is the NDT80 gene, a transcriptional regulator of middle meiotic gene expression required for the first division.

DNA-Binding Proteins↗

PubMed2003

Properties of natural double-strand-break sites at a recombination hotspot in Saccharomyces cerevisiae.

This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3'-to-5' conversion gradient, and two DSB sites located approximately 550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation.

Chromosomes↗