Suppression of a DNA polymerase delta mutation by the absence of the high mobility group protein Hmo1 in Saccharomyces cerevisiae.

The deletion of the gene encoding the high mobility group protein Hmo1 suppresses the growth retardation of the DNA pol delta mutation, pol3-14, at the restrictive temperature. pol3-14 mutant cells undergo cell cycle arrest, and hmo1Delta alleviates the arrest permitting continual division of the double mutant. Bypass of cell cycle control occurs with an increased rate of mutation. Both pol3-14 and hmo1Delta are mutators and their combination provokes a synergistic rate of CAN1 mutations. RAD18 controls branches of DNA repair pathways and its deletion also suppresses pol3 mutations. Comparing hmo1Delta and rad18Delta suppression of pol3-14 shows that while both require the presence of RAD52-mediated repair, their suppression is independent in that both can suppress in the presence of the other. We conclude that hmo1Delta suppression of pol3-14 occurs by a mechanism whereby normal controls on DNA integrity are breached and lesions flow into RAD52-mediated repair and error-prone pathways.

Interactions among DNA ligase I, the flap endonuclease and proliferating cell nuclear antigen in the expansion and contraction of CAG repeat tracts in yeast.

Among replication mutations that destabilize CAG repeat tracts, mutations of RAD27, encoding the flap endonuclease, and CDC9, encoding DNA ligase I, increase the incidence of repeat tract expansions to the greatest extent. Both enzymes bind to proliferating cell nuclear antigen (PCNA). To understand whether weakening their interactions leads to CAG repeat tract expansions, we have employed alleles named rad27-p and cdc9-p that have orthologous alterations in their respective PCNA interaction peptide (PIP) box. Also, we employed the PCNA allele pol30-90, which has changes within its hydrophobic pocket that interact with the PIP box. All three alleles destabilize a long CAG repeat tract and yield more tract contractions than expansions. Combining rad27-p with cdc9-p increases the expansion frequency above the sum of the numbers recorded in the individual mutants. A similar additive increase in tract expansions occurs in the rad27-p pol30-90 double mutant but not in the cdc9-p pol30-90 double mutant. The frequency of contractions rises in all three double mutants to nearly the same extent. These results suggest that PCNA mediates the entry of the flap endonuclease and DNA ligase I into the process of Okazaki fragment joining, and this ordered entry is necessary to prevent CAG repeat tract expansions.

Investigation of the stability of yeast rad52 mutant proteins uncovers post-translational and transcriptional regulation of Rad52p.

We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations--one a deletion of amino acids 210-327 and the other a missense mutation of residue 235--increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

C-terminal flap endonuclease (rad27) mutations: lethal interactions with a DNA ligase I mutation (cdc9-p) and suppression by proliferating cell nuclear antigen (POL30) in Saccharomyces cerevisiae.

During lagging-strand DNA replication in eukaryotic cells primers are removed from Okazaki fragments by the flap endonuclease and DNA ligase I joins nascent fragments. Both enzymes are brought to the replication fork by the sliding clamp proliferating cell nuclear antigen (PCNA). To understand the relationship among these three components, we have carried out a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its PCNA interaction domain. We recovered the flap endonuclease mutation rad27-K325* with a stop codon at residue 325. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. rad27-pX8 is temperature lethal and rad27-A358* grows slowly in combination with cdc9-p. Tests of mutation avoidance, DNA repair, and compatibility with DNA repair mutations showed that rad27-K325* confers severe phenotypes similar to rad27Delta, rad27-A358* confers mild phenotypes, and rad27-pX8 confers phenotypes intermediate between the other two alleles. High-copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Delta. These studies show the importance of the C terminus of the flap endonuclease in DNA replication and repair and, by virtue of the initial screen, show that this portion of the enzyme helps coordinate the entry of DNA ligase during Okazaki fragment maturation.

A high mobility group protein binds to long CAG repeat tracts and establishes their chromatin organization in Saccharomyces cerevisiae.

Long CAG repeat tracts cause human hereditary neurodegenerative diseases and have a propensity to expand during parental passage. Unusual physical properties of CAG repeat tracts are thought to contribute to their instability. We investigated whether their unusual properties alter the organization of CAG repeat tract chromatin. We report that CAG repeat tracts, embedded in yeast chromosomes, have a noncanonical chromatin organization. Digestion of chromatin with the restriction enzyme Fnu4HI reveals hypersensitive sites occurring approximately 125 bp apart in the repeat tract. To determine whether a non-histone protein establishes this pattern, we performed a yeast one-hybrid screen using CAG repeat tracts embedded in front of two reporter genes. The screen identified the high mobility group box protein Hmo1. Chromatin immunoprecipitation of epitope-tagged Hmo1 selectively precipitates CAG repeat tracts DNAs that range from 26 to 126 repeat units. Moreover, deletion of HMO1 drastically alters the Fnu4HI digestion pattern of CAG repeat chromatin. These results show that Hmo1 binds to CAG repeat tracts in vivo and establish the basis of their novel chromatin organization.