Coordinated regulation of XPA stability by ATR and HERC2 during nucleotide excision repair.

ATR (ATM and Rad3-related) is an essential regulator of the nucleotide excision repair (NER) mechanism. For NER activation, ATR phosphorylates XPA, the rate-limiting factor in the NER pathway. However, the role of XPA phosphorylation at serine 196 by ATR has been elusive. Here we show that ATR-mediated XPA phosphorylation enhances XPA stability by inhibiting HERC2-mediated ubiquitination and subsequent degradation. We analyzed stabilization of XPA with substitutions of Ser 196 either to aspartate (S196D), a phosphomimetic mutation, or to alanine (S196A), a phosphodeficient mutation. Upon ultraviolet damage, ATR facilitated HERC2 dissociation from the XPA complex to induce XPA stabilization. However, this regulation was abrogated in S196A-complemented XPA-deficient cells due to persistent association of HERC2 with this XPA complex, resulting in enhanced ubiquitination of S196A. Conversely, the S196D substitution showed delayed degradation kinetics compared with the wild-type and less binding with HERC2, resulting in reduced ubiquitination of S196D. We also found that XPA phosphorylation enhanced the chromatin retention of XPA, the interaction with its binding partners following DNA damage. Taken together, our study presents a novel control mechanism in the NER pathway by regulating the steady-state level of XPA through posttranslational modifications by which ATR-mediated phosphorylation induces XPA stabilization by antagonizing HERC2-catalyzed XPA ubiquitination.

Purification of circular YACs from yeast cells for DNA sequencing.

We describe a method for the purification of circular yeast artificial chromosome (YAC) DNA 120-150 kilobases (kb) in size that is of sufficient quantity and quality for restriction enzyme analysis and DNA sequencing. This method preferentially enriches for circular YAC DNA and avoids the time-consuming step of centrifugation in CsCl--ethidium bromide (EtBr) gradients. We applied this method to the purification of circular YACs carrying DNA segments that are extremely unstable in E. coli, including those that correspond to GAP2 and GAP3 on human chromosome 19. We showed that YAC DNA (GAP2 and GAP3) purified using this new method is clearly resolved in EtBr-stained gels. The sequence of YAC-GAP3 was obtained, representing the first GAP clone sequenced in YAC form. At present, it is estimated that there are more than 1000 gaps in the human genome that cannot be cloned using bacterial vectors. Thus, our new method may be very useful for completing the last stage of the human genome project.

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning.

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was >/=60 bp were utilized, approximately 2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is approximately 60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

An improved method for quantitative sugar analysis of glycoproteins.

Although there are numerous methods available to hydrolyze glycans utilizing strong acids, it all requires lengthy steps to obtain quantitative yield. We have developed a new simple one-step method for analysis of amino and neutral monosaccharides of glycoproteins quantitatively. Free monosaccharides were found to be stable during hydrolysis of glycans with 6 N HCI at 80 degrees C up to 2 h. Using this condition, analysis of free monosaccharides hydrolyzed from the bovine fetuin showed sugar composition of Gal: Man: GlcN: GaIN = 13.2: 11.0: 15.5: 2.6, which is closely matched with the reported value of 12.4: 9.6: 17.2: 2.7 (Townsend et al., ABRF News 8: 14, 1997). This method was shown to be applicable to varieties of well-characterized glycoproteins, erythropoietin, fibrinogen and soybean agglutinin. The amounts of sugars released under the condition were very close to the experimental values by other procedures or to the theoretical ones. This condition was found to be suitable for direct sugar analysis of fetuin, which have been immobilized onto polyvinylidene difluoride membrane. Based on these results, it support that the 6 N HCl/80 degrees C/2 h is the simplest method for quantitative analysis of monosaccharide composition of glycoproteins.

Differential organization and transcription of the cat2 gene cluster in aniline-assimilating Acinetobacter lwoffii K24.

CatABC genes encode proteins that are responsible for the first three steps of one branch of the beta-ketoadipate pathway involved in the degradation of various aromatic compound by bacteria. Aniline-assimilating Acinetobacter lwoffii K24 is known to have the two-catABC gene clusters (cat1 and cat2) on the chromosome (Kim et al., J. Bacteriol., 179: 5226-5231, 1997). The order of the cat2 gene cluster is catB2A2C2, which has not been found in other bacteria. In this report, we analyzed the transcriptional pattern of the cat2 gene cluster and completely sequenced a 5.8 kbp fragment containing the compactly clustered catB2A2C2 genes and four ORFs. Similar to the ORF(R1) of the cat1 gene cluster, an ORF highly homologous with the catR gene was found 102 by upstream of the catB2 gene and was designated as ORF(R2). Three ORFs, one putative reductase component (ORF(X2)) and two putative LysR family regulatory proteins (ORF(Y2), ORF(Z2)) were located next to the catC2 gene in the opposite direction of the cat2 gene cluster. Two ORFs, ORF(X2) and ORF(Y2), were significantly homologous with tdnB and tdnR of the aniline oxygenase complex of Pseudomonas putida UCC22. RT-PCR analysis and Northern blotting revealed that the catB2 gene is independently transcribed and that the catA2C2 genes are cotranscribed. A primer extension assay revealed that transcription of the catA2C2 gene starts in the C-terminal region of the catB2 gene. These results suggest that the cat2 gene cluster may be under a different gene adaptation from other cat gene clusters.

Meiotic role of SWI6 in Saccharomyces cerevisiae.

The transcript levels of DNA replication genes and some recombination genes in Saccharomyces cerevisiae fluctuate and peak at the G1/S boundary in the mitotic cell cycle. This fluctuation is regulated by MCB (Mlu I cell cycle box) elements which are bound by the DSC1/MBF1 complex consisting of Swi6 and Mbp1. It is also known that some of the MCB-regulated genes are induced by treatment with DNA damaging agents and in meiosis. In this report, the function of SWI6 in meiosis was investigated. Delta swi6 cells underwent sporulation as did wild-type cells. However, the deletion mutant cells showed reduced spore viability and lower frequency of recombination. The transcript levels of the recombination genes RAD51 and RAD54 , which have MCB elements, were reduced in Delta swi6 cells. The transcript levels of SWI6 itself were also induced and declined in meiosis. Furthermore, an increased dosage of SWI6 enhanced the transcript level of the RAD51 gene and also the recombination frequency in meiosis. These results suggest that SWI6 enhances the expression level of the recombination genes in meiosis in a dosage-dependent manner, which results in an effect on the frequency of meiotic recombination.

Phosphatidic acid-induced elevation of intracellular Ca2+ is mediated by RhoA and H2O2 in Rat-2 fibroblasts.

We have investigated possible roles of RhoA and H2O2 in the elevation of intracellular Ca2+ ([Ca2+]i) by phosphatidic acid (PA) in Rat-2 fibroblasts. PA induced a transient elevation of [Ca2+]i in the presence or absence of EGTA. Lysophosphatidic acid (LPA) also increased [Ca2+]i, but the sustained Ca2+ response was inhibited by EGTA. LPA stimulated the production of inositol phosphates, but PA did not. In the presence of EGTA, preincubation with LPA completely blocked the subsequent elevation of [Ca2+]i by PA, but not vice versa. PA stimulated the translocation of RhoA to the particulate fraction as did LPA. Scrape loading of C3 transferase inhibited the transient Ca2+ response to PA, but not to LPA, suggesting an essential role of RhoA in the elevation of [Ca2+]i by PA. H2O2 also induced a transient increase of [Ca2+]i as did PA. H2O2 scavengers, catalase and N-acetyl-L-cysteine, completely blocked the rise of [Ca2+]i stimulated by PA, but not by LPA. Furthermore, preincubation with PA blocked the subsequent Ca2+ response to H2O2, and the incubation with H2O2 also blocked the PA-induced rise of [Ca2+]i. Thus, it was suggested that PA stimulated Ca2+ release from PA-sensitive, but not inositol 1,4,5-trisphosphate-sensitive, Ca2+ stores by the activation of RhoA and intracellular H2O2.

Organization and transcriptional characterization of the cat1 gene cluster in Acinetobacter lwoffi K24.

Previously, we have reported that two clustered cat genes from Acenitobacter lwoffi K24 had different arrangements, catB1C1A1 and catB2A2C2 (Kim, S.I., S.-H. Leem, J.-S. Choi, Y.H. Chung, S. Kim, Y.-M. Park, Y.K. Park, Y.N. Lee, and K.-S. Ha. 1997, J. Bacteriol. 179, 5226-5231). By further analysis of the organization of the cat1 gene cluster, we obtained a complete sequence of the catB1 gene, which encoded 40.8-kDa polypeptide containing 379 amino acids, and found a open reading frame (ORF) coding a putative regulatory protein in upstream region of catB1 on plasmid pCD1-1. This ORF encoded 34.2-kDa polypeptide containing 379 amino acids and had more than 40% identity with catR, LysR family regulatory protein of Pseudomonas putida. RT-PCR, Northern blot analysis and primer extension assay for transcriptional analysis of the cat1 gene cluster revealed that the catB1C1 genes were cotranscribed and the catA1 gene was independently transcribed.

Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

Two novel type I catechol 1,2-dioxygenases inducible on aniline media were isolated from Acinetobacter lwoffii K24. Although the two purified enzymes, CD I1 and CD I2, had similar intradiol cleavage activities, they showed different substrate specificities for catechol analogs, physicochemical properties, and amino acid sequences. Two catA genes, catA1 and catA2, encoding by CD I1 and CD I2, respectively, were isolated from the A. lwoffii K24 genomic library by using colony hybridization and PCR. Two DNA fragments containing the catA1 and catA2 genes were located on separate regions of the chromosome. They contained open reading frames encoding 33.4- and 30.4-kDa proteins. The amino acid sequences of the two proteins matched well with previously determined sequences. Interestingly, further analysis of the two DNA fragments revealed the locations of the catB and catC genes as well. Moreover, the DNA fragment containing catA1 had a cluster of genes in the order catB1-catC1-catA1 while the catB2-catA2-catC2 arrangement was found in the catA2 DNA fragment. These results may provide an explanation of the different substrate specificities and physicochemical properties of CD I1 and CD I2.

Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.

Functions of the yeast meiotic recombination genes, MRE11 and MRE2.

Mutants defective in meiotic recombination were isolated using a disomic haploid strain of S. cerevisiae, and were classified into 11 genes. Two, MRE2 and MRE11, are new genes and nine are previously identified genes. The mre2 and mre11 deletion mutants are proficient in mitotic recombination, but are defective in meiotic recombination and in formation of viable spores. The spore inviability, however, is alleviated by an additional mutation, spo13, which bypasses meiosis I. In addition, neither meiosis specific DSBs at recombination hot-spots nor formation of synaptonemal complex occur in either mutant. Therefore, these two genes are involved in the formation of DSBs in meiotic recombination. While a temperature sensitive mre11-1 mutant is able to form DSBs at a permissive temperature, the formed DSBs are unable to resect at non permissive temperature. Therefore, the MRE11 gene is also involved in some step of the repair process after the DSB formation. Analysis of properties of the mre11 disruption mutant as well as the xrs2 mutant showed a similarity to those of the rad50 disruptant. We found that the mre11 disruption mutation is epistatic to rad50S mutation, as the xrs2 deletion mutation is epistatic to rad50S with regard to DSBs. Therefore, these three genes form an epistatic group. Interaction of the Mre11 protein with the Rad50 and the Xrs2 protein as well as alone was shown in vivo using the two-hybrid system. The MRE2 gene encodes a protein containing two sets of RRM. Deficiency of recombination in a mre2 mutant that has an amino acid substitution in the N-terminal RRM can be suppressed by the MER2 gene on the multicopy plasmid. Further analysis showed that the Mre2 protein is involved in meiosis-specific splicing of the MER2 transcripts in cooperation with the Mer1 protein. In conclusion, MRE genes are involved in the initiation of meiotic recombination through the formation of DSBs at recombination hot-spots in S. cerevisiae.

Hac1: a novel yeast bZIP protein binding to the CRE motif is a multicopy suppressor for cdc10 mutant of Schizosaccharomyces pombe.

We cloned by phenotypic complementation a novel Saccharomyces cerevisiae's multicopy suppressor of the Schizosaccharomyces pombe cdc10-129 mutant which we call HAC1, an acronym of 'homologous to ATF/CREB 1'. It encodes a bZIP (basic-leucine zipper) protein of 230 amino acids with close homology to the mammalian ATF/CREB transcription factor and gel-retardation assays showed that it binds specifically to the CRE motif. HAC1 is not essential for viability. However, the hac1 disruptant becomes caffeine sensitive, which is suppressed by multicopy expression of the yeast PDE2 (Phosphodiesterase 2) gene. Although the mRNA level of HAC1 is almost constitutive throughout the cell cycle, it fluctuates during meiosis. The upstream region of the HAC1 gene contains a T4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. These results suggest that HAC1 may also be one of the meiotic genes.

The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae.

Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.

Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae.

Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III. The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group. All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores. The spores are, however, not viable. The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination. The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene. By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis. Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed.

The MRE4 gene encodes a novel protein kinase homologue required for meiotic recombination in Saccharomyces cerevisiae.

The MRE4 gene was cloned by complementation of the defects of meiotic recombination and haploidization in an mre4-1 mutant. Disruption of MRE4 resulted in reduced meiotic recombination and spore inviability. The mre4 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass the reductional division. Analysis of meiotic DNA extracted from the mre4 mutant cells revealed that double-strand breaks occurred at the two sites of the HIS4-LEU2 recombination hot spot, but at a frequency of about 10-20% of the wild type. Northern blot analysis indicated that the MRE4 gene produces four transcripts of 1.63, 3.2, 4.0 and 6.2 kb. All of these transcripts are absent from mitotic cells and are meiotically induced. The DNA sequence of the MRE4 open reading frame predicts a 497-amino acids protein with a molecular mass of 56.8 kDa. The Mre4 protein contains highly conserved amino acid sequences found specifically in serine-threonine protein kinases. These results suggest that protein phosphorylation is required directly or indirectly for meiotic recombination.