Jourica A. Brandsma

Spt4 modulates Rad26 requirement in transcription‐coupled nucleotide excision repair

The nucleotide excision repair machinery can be targeted preferentially to lesions in transcribed sequences. This mode of DNA repair is referred to as transcription‐coupled repair (TCR). In yeast, the Rad26 protein, which is the counterpart of the human Cockayne syndrome B protein, is implicated specifically in TCR. In a yeast strain genetically deprived of global genome repair, a deletion of RAD26 renders cells UV sensitive and displays a defect in TCR. Using a genome‐wide mutagenesis approach, we found that deletion of the SPT4 gene suppresses the rad26 defect. We show that suppression by the absence of Spt4 is specific for a rad26 defect and is caused by reactivation of TCR in a Rad26‐independent manner. Spt4 is involved in the regulation of transcription elongation. The absence of this regulation leads to transcription that is intrinsically competent for TCR. Our findings suggest that Rad26 acts as an elongation factor rendering transcription TCR competent and that its requirement can be modulated by Spt4.

Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe.

The RAD52 gene of Saccharomyces cerevisiae is essential for repair of DNA double-strand breaks (DSBs) by homologous recombination. Inactivation of this gene confers hypersensitivity to DSB-inducing agents and defects in most forms of recombination. The rad22+ gene in Schizosaccharomyces pombe (here referred to as rad22A+) has been characterized as a homolog of RAD52 in fission yeast. Here, we report the identification of a second RAD52 homolog in Schizosaccharomyces pombe, called rad22B+. The amino acid sequences of Rad22A and Rad22B show significant conservation (38% identity). Deletion mutants of respectively, rad22A and rad22B, show different phenotypes with respect to sensitivity to X-rays and the ability to perform homologous recombination as measured by the integration of plasmid DNA. Inactivation of rad22A+ leads to a severe sensitivity to X-rays and a strong decrease in recombination (13-fold), while the rad22B mutation does not result in a decrease in homologous recombination or a change in radiation sensitivity. In a rad22A-rad22B double mutant the radiation sensitivity is further enhanced in comparison with the rad22A single mutant. Overexpression of the rad22B+ gene results in partial suppression of the DNA repair defects of the rad22A mutant strain. Meiotic recombination and spore viability are only slightly affected in either single mutant, but outgrowth of viable spores is almost 31-fold reduced in the rad22A-rad22B double mutant. The results obtained imply a crucial role for rad22A+ in repair and recombination in vegetative cells just like RAD52 in S. cerevisiae. The rad22B+ gene presumably has an auxiliary role in the repair of DSBs. The drastic reduced spore viability in the double mutant suggests that meiosis in S. pombe is dependent on the presence of either rad22A+ or rad22B+.

The first zinc-binding domain of UvrA is not essential for UvrABC-mediated DNA excision repair

Specific mutations in uvrA were introduced to analyze the role of the zinc-binding domains of the protein in DNA excision repair. Zinc-coordinating cysteines were substituted into non-coordinating serine or glycine residues. Mutations leading to changes in the second zinc-binding domain had a profound effect on UV survival in vivo; however these mutant proteins could not be isolated for in vitro analyses. Amino acid substitutions in the first zinc-binding domain had very little effect on UV survival in vivo. In vitro analyses showed that although this domain no longer coordinates zinc, ATPase activity, helicase activity, DNA binding, incision of damaged DNA and DNA repair synthesis appeared to be normal. Therefore it seems that the first zinc-binding domain of UvrA is not essential for DNA excision repair.

Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Eschenchia coli K-12

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation achieved when the ssb gene us inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.

The NER protein Rad33 shows functional homology to human Centrin2 and is involved in modification of Rad4

In the yeast Saccharomyces cerevisiae the Rad4-Rad23 complex is implicated in the initial damage recognition of the Nucleotide Excision Repair (NER) pathway. NER removes a variety of lesions via two subpathways: Transcription Coupled Repair (TCR) and Global Genome Repair (GGR). We previously showed that the new NER protein Rad33 is involved in both NER subpathways TCR and GGR. In the present study we show UV induced modification of Rad4 that is strongly increased in cells deleted for RAD33. Modification of Rad4 in rad33 cells does not require the incision reaction but is dependent on the TCR factor Rad26. The predicted structure of Rad33 shows resemblance to the Centrin homologue Cdc31. In human cells, Centrin2 binds to XPC and is involved in NER. We demonstrate that Rad4 binds Rad33 directly and via the same conserved amino acids required for the interaction of XPC with Centrin2. Disruption of the Rad4-Rad33 interaction is sufficient to enhance the modification of Rad4 and results in a repair defect similar to that of a rad33 mutant. The current study suggests that the role of Rad33 in the Rad4-Rad23 complex might have parallels with the role of Centrin2 in the XPC-HHR23B complex.

The Rad4 homologue YDR314C is essential for strand‐specific repair of RNA polymerase I‐transcribed rDNA in Saccharomyces cerevisiae

The Saccharomyces cerevisiae protein Rad4 is involved in damage recognition in nucleotide excision repair (NER). In RNA polymerase II‐transcribed regions Rad4 is essential for both NER subpathways global genome repair (GGR) and transcription coupled repair (TCR). In ribosomal DNA (rDNA), however, the RNA polymerase I‐transcribed strand can be repaired in the absence of Rad4. In Saccharomyces cerevisiae the YDR314C protein shows homology to Rad4. The possible involvement of YDR314C in NER was studied by analysing strand‐specific cyclobutane pyrimidine dimer (CPD) removal in both RNA pol I‐ and RNA pol II‐transcribed genes. Here we show that the Rad4‐independent repair of rDNA is dependent on YDR314C. Moreover, in Rad4 proficient cells preferential repair of the transcribed strand of RNA pol I‐transcribed genes was lost after deletion of YDR314C, demonstrating that Rad4 cannot replace YDR314C. CPD removal from the RNA pol II‐transcribed RPB2 gene was unaffected in ydr314c mutants. We conclude that the two homologous proteins Rad4 and YDR314C are both involved in NER and probably have a similar function, but operate at different loci in the genome and are unable to replace each other.

Cloning of Schizosaccharomyces pombe rph16+, a gene homologous to the Saccharomyces cerevisiae RAD16 gene.

The RAD16 gene is involved in the nucleotide excision repair of UV damage in the transcriptional silenced mating type loci (Terleth et al., 1990 and Bang et al., 1992) and in non-transcribed stands of active genes in Saccharomyces cerevisiae (Verhage et al., 1994). Using touchdown-PCR with primers derived from various domains of the S. cerevisiae Rad 16 protein, a specific Schizosaccharomyces pombe probe was isolated. This probe was used to obtain the complete RAD16 homologous gene from a S. pombe chromosomal bank. DNA sequence analysis of the rph16+ gene revealed an open reading frame of 854 amino acids. Comparison of the amino acid sequences of the Rhp16 and Rad16 proteins showed a high level of conservation: 68% similarity. The Rhp16 protein sequence contains the two Zn-finger motifs and the putative helicase domains as found in the Rad16 protein. Like the RAD16, the rph16+ gene is UV-inducible (Bang et al., 1995). In analogy with the rad16 mutant, the rhp16 disruption mutant is viable and grows normally, indicating that the gene does not have an essential function. The rhp16 disruption mutant is not sensitive for UV but is sensitive for cisplatin. The rhp16+ gene cloned behind the GAI 1 promoter partially complements the UV sensitivity and the defect in the non-transcribed strand DNA repair of a S. cerevisiae rad16 mutant, indicating functional homology between the rhp16+ and RAD16 genes. The structural and functional homology between the two genes suggests that the RAD16 dependent subpathway of NER for the repair of non-transcribed DNA is evolutionary conserved.

Location and cloning of the ultraviolet-sensitizing function from the chromosomally associated IncJ group plasmid, R391.

The IncJ plasmid R391, which specifies a uv-sensitizing function, has been shown to be associated with chromosomal DNA. Deletions originating from Tn10 insertion into the kanamycin-resistance determinant of plasmid R391 gave rise to uv-resistant derivatives. This apparent linkage between the kanamycin-resistance determinant and the uv-sensitizing gene(s) was used to clone the uv-sensitizing function from plasmid R391 into pUR222. A recombinant plasmid containing both functions (KanR and Uvs+) was obtained. The uv-sensitizing function was mapped to a 4-kb EcoRI fragment.

The in vitro more efficiently repaired cisplatin adduct cis-Pt.GG is in vivo a more mutagenic lesion than the relative slowly repaired cis-Pt.GCG adduct

The toxic effect and the mutagenicity of two differentially repaired site-specific cis-diamminedichloroplatinum(II) (cis-DDP) lesions were investigated. Detailed analysis of the UvrABC-dependent repair of the two lesions in vitro showed a more efficient repair of the cis-Pt.GG adduct compared to that of the cis-Pt.GCG adduct (Visse et al., 1994). Furthermore, previously, a dependency of cis-DDP mutagenesis on UvrA and UvrB, but not on UvrC was found (Brouwer et al., 1988). To possibly relate survival and mutagenesis to repair, plasmids containing the same site-specific cis-DDP lesions as those that were used in the detailed repair studies were transformed into Escherichia coli. The results indicate that both lesions are very efficiently bypassed in vivo. Mutation analysis was performed using a denaturing gradient gel electrophoresis technique, which allows identification of mutations without previous selection. Although the cis-Pt.GG adduct is in vitro more efficiently repaired than the cis-Pt.GCG adduct, it appeared to be more mutagenic. We present a model in which this result is related to the previously observed dependency of the mutagenicity of cis-DDP lesions on the Uvr A and B proteins.

Plasmids Carrying the uvrA and uvrC Genes of Escherichia coli K12: Construction and Properties

From the Carbon-Clarke collection, a plasmid harbouring the uvrC gene was identified (pLC13–12) and subsequently subcloned into pBR322. The UvrC+ plasmid pCA32 complemented UvrC deficient bacteria to the wild type phenotype. The uvrC gene has been assigned to a 2,000 bp BgIII DNA fragment. Upon introduction into minicells, pCA32 directs the synthesis of a protein of 28,000 dalton, which most likely is encoded by the uvrC gene.A novel method is described for the cloning of genes, for which direct selection is impracticable. The technique has been employed in the construction of a plasmid carrying lexA, uvrA, ssb on pACYC184.