The role of genetic toxicology in drug discovery and optimization.

Genetic toxicology data is used as a surrogate for long-term carcinogenicity data during early drug development. The aim of genotoxicity testing is to identify potentially hazardous drug candidates. Results from genetic toxicology tests in combination with acute and subchronic animal data are used as the basis to approve clinical trials of drug candidates. With few exceptions, mutagenic compounds are dropped from development and clastogenic compounds result in unfavorable labeling, require disclosure in clinical trial consent forms, and can impact the marketability of a new drug. Therefore, genetic toxicology testing in drug discovery and optimization serves to quickly identify mutagens and remove them from development. Additionally, clastogenicity can delay drug development by requiring additional testing to determine in vivo relevance of in vitro clastogenic responses. Clastogenicity screening is conducted so any additional testing can be planned and perhaps integrated into other toxicity studies to expedite progression of drugs into the clinic. Commercially available genotoxicity and carcinogenicity predictive software systems used for decision support by ICSAS, FDA/CDER is described along with the strengths and weakness of each system. The FDA has concentrated on using a consensus approach to maximize certainty for positive predictions at the expense of sensitivity. The consensus approach consists of requiring 2 complementary software packages, such as MC4PC and MDL QSAR models, to agree that a compound has a genotoxic or carcinogenic liability. Mutagenicity and clastogenicity screening tests are described along with advantages and disadvantages of each test. Several testing strategies are presented for consideration.

The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development.

Nucleotide excision repair in Arabidopsis thaliana differs from other eukaryotes as it contains two paralogous copies of the corresponding XPB/RAD25 gene. In this work, the functional characterization of one copy, AtXPB1, is presented. The plant gene was able to partially complement the UV sensitivity of a yeast rad25 mutant strain, thus confirming its involvement in nucleotide excision repair. The biological role of AtXPB1 protein in A. thaliana was further ascertained by obtaining a homozygous mutant plant containing the AtXPB1 genomic sequence interrupted by a T-DNA insertion. The 3' end of the mutant gene is disrupted, generating the expression of a truncated mRNA molecule. Despite the normal morphology, the mutant plants presented developmental delay, lower seed viability and a loss of germination synchrony. These plants also manifested increased sensitivity to continuous exposure to the alkylating agent MMS, thus suggesting inefficient DNA damage removal. These results indicate that, although the duplication seems to be recent, the features described for the mutant plant imply some functional or timing expression divergence between the paralogous AtXPB genes. The AtXPB1 protein function in nucleotide excision repair is probably required for the removal of lesions during seed storage, germination and early plant development.

Deletion of the CSB homolog, RAD26, yields Spt(-) strains with proficient transcription-coupled repair.

It has been previously shown that disruption of RAD26 in yeast strain W303-1B results in a strain that is deficient in transcription-coupled repair (TCR), the preferential repair of the transcribed strand of an expressed gene over the non-transcribed strand and the rest of the genome. RAD26 encodes a protein that is homologous to Cockayne syndrome group B protein (CSB) and is a member of the SWI2/SNF2 family of DNA-dependent ATPases involved in chromatin remodeling. Like the rad26 mutant, cells from Cockayne syndrome patients are defective in TCR. We examined the role of Rad26 in TCR by disrupting RAD26 in two repair-proficient laboratory strains and, remarkably, observed no effect upon TCR. Our results indicate that disruption of RAD26 alone is insufficient to impair TCR. Thus, W303-1B must already possess a mutation that, together with disruption of RAD26, causes a deficiency in TCR. We suggest that other genes are mutated in Cockayne syndrome cells that contribute to the deficiency in TCR. Surprisingly, deletion of RAD26 results in expression of genes that are repressed by flanking transposon delta elements, an Spt(-) phenotype. The delta elements appear to perturb local chromatin structure. Expression of genes flanked by delta elements in rad26Delta mutants is consistent with a role for Rad26 in chromatin remodeling.

Transcription-coupled repair in yeast is independent from ubiquitylation of RNA pol II: implications for Cockayne's syndrome.

Cockayne's syndrome cells lack transcription-coupled nucleotide excision repair (TCR) and ubiquitylation of RNA polymerase II large subunit (RNA pol II LS), suggesting that ubiquitylation of RNA pol II LS may be necessary for TCR in eukaryotes. Rsp5 is the sole yeast ubiquitin-protein ligase that ubiquitylates RNA pol II LS in cells exposed to DNA-damaging agents. In yeast lacking functional Rsp5, there is no ubiquitylation of RNA pol II LS. We show here that removal, repression, or over-expression of Rsp5 has no effect on TCR, demonstrating that ubiquitylation of the RNA pol II LS is not required for TCR. We infer that the lack of ubiquitylation of RNA pol II LS in Cockayne's syndrome cells does not cause their defect in TCR.

Transcription-coupled DNA repair in yeast transcription factor IIE (TFIIE) mutants.

We examined the role of yeast transcription initiation factor IIE (TFIIE) in eukaryotic transcription-coupled repair (TCR), the preferential removal of DNA damage from the transcribed strands of genes over non-transcribed sequences. TFIIE can recruit the transcription initiation/repair factor TFIIH to the RNA polymerase II (RNA pol II) initiation complex to facilitate promoter clearance. Following exposure to UV radiation, the RNA pol II elongation complex is blocked at sites of UV-induced DNA damage, and may be recognized by nucleotide excision repair proteins, thus enabling TCR. The TFA1 gene encodes the large subunit of TFIIE. We determined how DNA repair is affected by TFA1 conditional mutations. In particular, we find proficient TCR in a heat-sensitive tfa1 mutant at the non-permissive temperature during which growth is inhibited and overall RNA pol II transcription is reported to be inhibited. We demonstrate that transcription of the RPB2 gene was reduced, but readily detectable, in the heat-sensitive tfa1 mutant at the non-permissive temperature and thereby prove that TCR does occur in an expressed gene in the absence of TFIIE in vivo. We demonstrate that TCR occurs even at low levels of transcription.

Mismatch repair mutants in yeast are not defective in transcription-coupled DNA repair of UV-induced DNA damage.

Transcription-coupled repair, the targeted repair of the transcribed strands of active genes, is defective in bacteria, yeast, and human cells carrying mutations in mfd, RAD26 and ERCC6, respectively. Other factors probably are also uniquely involved in transcription-repair coupling. Recently, a defect was described in transcription-coupled repair for Escherichia coli mismatch repair mutants and human tumor cell lines with mutations in mismatch repair genes. We examined removal of UV-induced DNA damage in yeast strains mutated in mismatch repair genes in an effort to confirm a defect in transcription-coupled repair in this system. In addition, we determined the contribution of the mismatch repair gene MSH2 to transcription-coupled repair in the absence of global genomic repair using rad7 delta mutants. We also determined whether the Rad26-independent transcription-coupled repair observed in rad26 delta and rad7 delta rad26 delta mutants depends on MSH2 by examining repair deficiencies of rad26 delta msh2 delta and rad7 delta rad26 delta msh2 delta mutants. We found no defects in transcription-coupled repair caused by mutations in the mismatch repair genes MSH2, MLH1, PMS1, and MSH3. Yeast appears to differ from bacteria and human cells in the capacity for transcription-coupled repair in a mismatch repair mutant background.

DNA repair deficiencies associated with mutations in genes encoding subunits of transcription initiation factor TFIIH in yeast.

Several proteins, including Rad3 and Rad25(Ssl2), are essential for nucleotide excision repair (NER) and function in the RNA polymerase II transcription initiation complex TFIIH. Mutations in genes encoding two other subunits of TFIIH, TFB1 and SSL1, result in UV sensitivity and have been shown to take part in NER in an in vitro system. However, a deficiency in global NER does not exclude the possibility that such repair-deficient mutants can perform transcription-coupled repair (TCR), as shown for xeroderma pigmentosum group C. To date, temperature-sensitive C-terminal truncations of Tfbl are the only TFIIH mutations that result in intermediate UV sensitivity, which might indicate a deficiency in either the global NER or TCR pathways. We have directly analyzed both TCR and global NER in these mutants. We found that ssl1, rad3 and tfb1 mutants, like rad25(ssl2-xp) mutants, are deficient in both the global NER and TCR pathways. Our results support the view that the mutations in any one of the genes encoding subunits of TFIIH result in deficiencies in both global and TCR pathways of NER. We suggest that when subunits of TFIIH are in limiting amounts, TCR may preclude global NER.

Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions.

The SNF2 family of proteins includes representatives from a variety of species with roles in cellular processes such as transcriptional regulation (e.g. MOT1, SNF2 and BRM), maintenance of chromosome stability during mitosis (e.g. lodestar) and various aspects of processing of DNA damage, including nucleotide excision repair (e.g. RAD16 and ERCC6), recombinational pathways (e.g. RAD54) and post-replication daughter strand gap repair (e.g. RAD5). This family also includes many proteins with no known function. To better characterize this family of proteins we have used molecular phylogenetic techniques to infer evolutionary relationships among the family members. We have divided the SNF2 family into multiple subfamilies, each of which represents what we propose to be a functionally and evolutionarily distinct group. We have then used the subfamily structure to predict the functions of some of the uncharacterized proteins in the SNF2 family. We discuss possible implications of this evolutionary analysis on the general properties and evolution of the SNF2 family.

Repair and transcription. Collision or collusion?

While some proteins have distinct responsibilities in both transcription and DNA repair, additional proteins are needed to couple these essential DNA transactions in expressed genes.

The COOH terminus of suppressor of stem loop (SSL2/RAD25) in yeast is essential for overall genomic excision repair and transcription-coupled repair.

We examined several yeast strains with different mutations in the essential SSL2 (Suppressor of Stem Loop, also called RAD25) gene for their ability to remove cyclobutane pyrimidine dimers from expressed genes, and from the genome overall. The SSL2 protein has a high degree of amino acid sequence identity to the protein encoded by the human ERCC3 gene (Gulyas, K. D., and Donahue, T. F. (1992) Cell 69, 1031-0142). The mutant allele SSL2-XP encodes a protein resembling the mutated ERCC3 protein from UV-sensitive human cells belonging to xeroderma pigmentosum complementation group B and Cockayne's syndrome (CS) complementation group C (Weeda, G., van Ham, R. C. A., Vermeulen, W., Bootsma, D., van der Eb, A. J., and Hoeijmakers, J. H. J. (1990) Cell 62, 777-791; Gulyas and Donahue, 1992). The SSL2-XP allele confers UV sensitivity on yeast strain KG119. We found that the biochemical basis for the UV sensitivity of KG119 is a complete deficiency in the removal of cyclobutane pyrimidine dimers from the overall genome as well as a deficiency in transcription-coupled repair. This is the first analysis of the DNA repair defect responsible for the UV sensitivity of cells carrying the SSL2-XP allele, and it documents the similarity of the defect to that associated with XP-B/CS-C, and the difference between this defect and that in cells belonging to CS complementation groups A and B.

Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription.

While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia coli, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARS1 plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpb1-1, in the largest subunit of RNA polymerase II. When exponentially growing rpb1-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpb1-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpb1-1 cells was reduced to that in the nontranscribed strand. These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast.

The gene encoding ARS-binding factor I is essential for the viability of yeast.

The gene encoding a yeast ARS-binding protein, ABF I, has been cloned by screening a genomic lambda gt11 library using monoclonal and polyclonal antibodies against ABF I. ABF I is of interest because it not only binds to ARSs but also to the 5'-flanking region of genes encoding proteins involved in transcription, translation, respiration, and cell-cycle control. The cloned gene has been used to prepare null mutants, which further demonstrate the importance of the ABF I protein by showing that it is essential for vegetative growth. ABF1 maps to chromosome V. The DNA sequence of the ABF1 gene reveals several motifs characteristic of DNA-binding proteins but shows no overall similarity to any protein of known function.

Purification and characterization of proteins that bind to yeast ARSs.

Two proteins that bind to yeast ARS DNA have been purified using conventional and oligonucleotide affinity chromatography. One protein has been purified to homogeneity and has a mass of 135 kDa. Competitive binding studies and DNase I footprinting show that the protein binds to a sequence about 80 base pairs away from the core consensus in the region known as domain B. This region has previously been shown to be required for efficient replication of plasmids carrying ARS1 elements. To investigate further whether the protein might have a function related to the ability of ARSs to act as replicators, binding to another ARS was tested. The protein binds to the functional ARS adjacent to the silent mating type locus HMR, called the HMR-E ARS, about 60 base pairs from the core consensus sequence. Surprisingly, there is little homology between the binding site at the HMR-E ARS and the binding site at ARS1. The 135-kDa protein is probably the same as ABF-I (SBF I) (Shore, D., Stillman, D. J. Brand, A. H., and Nasmyth, K. A. (1987) EMBO J. 6, 461-467; Buchman, A. R., Kimmerly, W. J., Rine, J., and Kornberg, R. D. (1988) Mol. Cell. Biol. 8, 210-225). A second DNA-binding protein was separated from ABF-I during later stages of the purification. This protein, which we designate ABF-III, also binds specifically to the ARS1 sequence, as shown by DNase I footprinting, at a site adjacent to the ABF-I recognition site. Purification of these two ARS binding proteins should aid in our understanding of the complex mechanisms that regulate eukaryotic DNA replication.