Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities.

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).

ENOD8, a novel early nodule-specific gene, is expressed in empty alfalfa nodules.

The alfalfa ENOD8 nodule-specific gene is expressed in empty nodules elicited by exopolysaccharide-deficient Rhizobium meliloti, and it is expressed early in nodule development. An ENOD8 cDNA was sequenced and found to encode a novel product. Its deduced polypeptide sequence was found to be similar to the nonproline-rich domains of the putative polypeptides encoded by a class of anther-specific genes from Arabidopsis thaliana and Brassica napus. The role of the ENOD8 gene product is predicted to be in nodule organogenesis. ENOD8 is expressed in a developmental pathway triggered as a result of Rhizobium nodulation signal transduction.

SCH9, a putative protein kinase from Saccharomyces cerevisiae, affects HOT1-stimulated recombination.

HOT1 is a mitotic recombination hotspot derived from yeast rDNA. To further study HOT1 function, trans-acting H OT1 recombination mutants (hrm) that alter hotspot activity were isolated. hrm2-1 mutants have decreased HOT1 activity and grow slowly. The HRM2 gene was cloned and found to be identical to SCH9, a gene that affects a growth-control mechanism that is partially redundant with the cAMP-dependent protein kinase A (PKA) pathway. Deletion of SCH9 decreases HOT1 and rDNA recombination but not other mitotic exchange. Although high levels of RNA polymerase I transcription initiated at HOT1 are required for its recombination-stimulating activity, sch9 mutations do not affect transcription initiated within HOT1. Thus, transcription is necessary but not sufficient for HOT1 activity. TPK1, which encodes a catalytic subunit of PKA, is a multicopy suppressor of the recombination and growth defects of sch9 mutants, suggesting that increased PKA activity compensates for SCH9 loss. RAS2( val19), which codes for a hyperactive RAS protein and increases PKA activity, suppresses both phenotypic defects of sch9 mutants. In contrast to TPK1 and RAS2(val19), the gene for split zinc finger protein 1 (SFP1) on a multicopy vector suppresses only the growth defects of sch9 mutants, indicating that growth and HOT1 functions of Sch9p are separable. Sch9p may affect signal transduction pathways which regulate proteins that are specifically required for HOT1-stimulated exchange.

Elimination of replication block protein Fob1 extends the life span of yeast mother cells.

A cause of aging in yeast is the accumulation of circular species of ribosomal DNA (rDNA) arising from the 100-200 tandemly repeated copies in the genome. We show here that mutation of the FOB1 gene slows the generation of these circles and thus extends life span. Fob1p is known to create a unidirectional block to replication forks in the rDNA. We show that Fob1p is a nucleolar protein, suggesting a direct involvement in the replication fork block. We propose that this block can trigger aging by causing chromosomal breaks, the repair of which results in the generation of rDNA circles. These findings may provide a novel link between metabolic rate and aging in yeast and, perhaps, higher organisms.