Biomedical subjects

L K Derr

Publications and source records attributed to L K Derr.

6 recordsLinked to original sources

PubMed1998

The involvement of cellular recombination and repair genes in RNA-mediated recombination in Saccharomyces cerevisiae.

We previously demonstrated that a reverse transcript of a cellular reporter gene (his3-AI) can serve as the donor for gene conversion of a chromosomal his3-deltaMscI target sequence, and that this process requires the yeast recombination gene RAD52. In this study, we examine the involvement of other recombination and repair genes in RNA-mediated recombination, and gain insight into the nature of the recombination intermediate. We find that mutation of the mitotic RecA homologs RAD51, RAD55, and RAD57 increases the rate of RNA-mediated recombination relative to the wild type, and that these gene functions are not required for RNA-mediated gene conversion. Interestingly, RAD1 is required for RNA-mediated gene conversion of chromosomal his3-deltaMscI sequences, suggesting that the cDNA intermediate has a region of nonhomology that must be removed during recombination with target sequences. The observation that both RAD1 and RAD52 are required for RNA-mediated gene conversion of chromosomal but not plasmid sequences indicates a clear difference between these two pathways of homologous RNA-mediated recombination.

DNA Repair↗

PubMed1993

A role for reverse transcripts in gene conversion.

Recombination between a diffusible reverse transcript and its homologous chromosomal allele has been proposed as a mechanism for the precise removal of introns from DNA and gene conversion of dispersed repeated sequences. We have reported that RNA-mediated recombination occurs in the yeast Saccharomyces cerevisiae. This recombination requires expression of the retrotransposon Ty, and results in intron loss from a plasmid-borne marker gene and the formation of pseudogenes. Because the pseudogenes are embedded in Ty sequences, chromosomal insertion could have been mediated by Ty integrase or by homologous recombination with endogenous Ty sequences. The structure of the chromosomal recombinants and the fact that plasmid and chromosomal recombination can have different requirements demanded a direct demonstration of RNA-mediated gene conversion of a chromosomal allele. Here we report the first demonstration, to our knowledge, of recombination between a reverse transcript and its chromosomal homologue and describe an assay that specifically detects this novel recombination pathway.

Base Sequence↗

PubMed1993

Recombination initiated by double-strand breaks.

The HO endonuclease was used to introduce a site-specific double-strand break (DSB) in an interval designed to monitor mitotic recombination. The interval included the trp1 and his3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Mitotic recombination was monitored in a diploid carrying heteroalleles of trp1 and his3. The normal recognition sites for the HO endonuclease were mutated at the MAT alleles and a synthetic recognition site for HO endonuclease was placed between trp1 and his3 on one of the chromosomes. HO-induced cleavage resulted in efficient recombination in this interval. Most of the data can be explained by double-strand gap repair in which the cut chromosome acts as the recipient. However, analysis of some of the recombinants indicates that regions of heteroduplex were generated flanking the site of the cut, and that some recombinants were the result of the cut chromosome acting as the genetic donor.

Base Sequence↗

PubMed1991

RNA-mediated recombination in S. cerevisiae.

The existence of pseudogenes and the observation of intron loss suggest that RNA can serve as an intermediate in recombination. We used a HIS3 reporter gene to show that RNA-mediated recombination occurs in yeast. His3+ prototroph formation required transcription and expression of the retrotransposon Ty. Two RNA-mediated recombination events were detected: homologous recombination between the cDNA and plasmid his3 sequences, resulting in intron loss, and insertion of the cDNA into the chromosome in the absence of HIS3 homology. The chromosomal His3+ prototrophs showed many hallmarks of naturally occurring pseudogenes. They integrated at novel sites in the chromosome, lacked introns, and possessed poly(A) tracts. Additionally, their 5' ends corresponded with the site of initiation of the GAL1 transcript.

Base Sequence↗

PubMed1990

Expression and function of the uvsW gene of bacteriophage T4.

The uvsW gene of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including replication, recombination and repair. To approach the function of uvsW, the structure and expression of the uvsW gene were first explored. Molecular analyses defined the promoter region, the transcriptional start site, and the probable initiation codon. The required promoter region contains a sequence resembling the consensus for T4 late promoters. Furthermore, transcriptional analyses indicated that uvsW is expressed as a late gene, providing a time frame for uvsW action. Several novel observations restrict possible models for uvsW function. A uvsW-deletion mutation reduced overall phage-phage recombination 1.7-fold, but reduced plasmid integration tenfold relative to the wild-type. Thus, the UsvW protein plays a critical role in a specific recombination pathway involving simple reciprocal exchange. One of the most intriguing phenotypes associated with uvsW mutations is the restoration of arrested DNA synthesis caused by mutations that block secondary initiation, the major mode by which replication initiates at late times in wild-type infections. Experiments with plasmid model systems indicate that a uvsW mutation does not restore the arrested DNA synthesis by rescuing secondary initiation directly. Rather, a uvsW mutation appears to allow some alternative mode of late replication, implying that the UvsW protein normally represses this alternative pathway. The rifampicin resistance of uvsW-repressed replication suggests that it involves either tertiary initiation or some novel mode of initiation. Finally, the inappropriate early expression of uvsW from a heterologous promoter blocks most early phage DNA synthesis in a uvsY-mutant infection, suggesting that the UvsW protein is normally the key regulatory factor in the switch from early to late DNA replication. According to this suggestion, the restored late replication in a uvsW mutant is an abnormal continuation of an early mode(s) of replication.

Amino Acid Sequence↗

PubMed1990

Isolation and genetic characterization of new uvsW alleles of bacteriophage T4.

The uvsW gene of bacteriophage T4 is required for wild-type levels of recombination, for normal survival and mutagenesis after UV irradiation, and for wild-type resistance to hydroxyurea. Additionally, uvsW mutations restore the arrested DNA synthesis caused by mutations in any of several genes that block secondary initiation (recombination-primed replication, the major mode of initiation at late times), but only partially restore the reduced burst size. A uvsW deletion mutation was constructed to establish the null-allele phenotype, which is similar but not identical to the phenotype of the canonical uvsW mutation, and to demonstrate convincingly that the uvsW gene is nonessential (although uvsW mutations severely compromise phage production). In an attempt to uncouple the diverse effects of uvsW mutations, temperature-sensitive uvsWts mutants were isolated. Recombination and replication effects were partially uncoupled in these mutants, suggesting distinct and separable roles for uvsW in the two processes. Furthermore, the restoration of DNA synthesis but not recombination in the double mutants uvsW uvsX and uvsW uvsY prompts the hypothesis that the restored DNA synthesis is not recombinationally initiated.

Alleles↗