Amna Butt

Flipping of alkylated DNA damage bridges base and nucleotide excision repair

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O6-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O6-methylguanine or cigarette-smoke-derived O6-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.

Chromatographic separations as a prelude to two-dimensional electrophoresis in proteomics analysis.

Current methods of proteome analysis rely almost solely on two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) followed by the excision of individual spots and protein identification using mass spectrometry (MS) and database searching. 2‐D PAGE is denaturing in both dimensions and, thus, cannot indicate functional associations between individual proteins. Moreover, less abundant proteins are difficult to identify. To simplify the proteome, and explore functional associations, nondenaturing anion exchange column chromatography was used to separate a soluble protein extract from Escherichia coli.Successive fractions were then analysed using 2‐D PAGE and selected spots from both the gels for the start material and the fractionated material were quantified and identified by peptide mass fingerprinting using a MALDI‐TOF mass spectrometer. Enrichments of up to 13‐fold were attained for individual protein spots and peptide mass fingerprints were of significantly higher quality after chromatographic separation. The marked anomalies between predicted p and column elution position contrasted with the almost perfect correlation with migration distance on isoelectric focusing (IEF) and were explored further for basic proteins.

A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics.

The identification of individual protein species within an organisms proteome has been optimised by increasing the information produced from mass spectral analysis through the chemical derivatisation of tryptic peptides and the development of new software tools. Peptide fragments are subjected to two forms of derivatisation. First, lysine residues are converted to homoarginine moieties by guanidination. This procedure has two advantages, first, it usually identifies the C‐terminal amino acid of the tryptic peptide and also greatly increases the total information content of the mass spectrum by improving the signal response of C‐terminal lysine fragments. Second, an Edman‐type phenylthiocarbamoyl (PTC) modification is carried out on the N‐terminal amino acid. The renders the first peptide bond highly susceptible to cleavage during mass spectrometry (MS) analysis and consequently allows the ready identification of the N‐terminal residue. The utility of the procedure has been demonstrated by developing novel bioinformatic tools to exploit the additional mass spectral data in the identification of proteome proteins from the yeast Saccharomyces cerevisiae. With this combination of novel chemistry and bioinformatics, it should be possible to identify unambiguously any yeast protein spot or band from either two‐dimensional or one‐dimensional electropheretograms.

A novel DNA damage recognition protein in Schizosaccharomyces pombe

Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.

Atl1 Regulates Choice between Global Genome and Transcription-Coupled Repair of O(6)-Alkylguanines.

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.

Functional Analysis of six novel ORFs on the left arm of Chromosome XII of Saccharomyces cerevisiae reveals three of them responding to S-starvation.

Six novel Open Reading Frames (ORFs) located on the left arm of the chromosome XII (YLL061w, YLL060c, YLL059c, YLL058w, YLL057c and YLL056c) have been analysed using either short‐flanking homology (SFH) or long‐flanking homology (LFH) gene replacement. Sporulation and tetrad analysis showed none of these ORFs to be essential for vegetative growth. The standard EUROFAN growth tests failed to reveal any obvious phenotypes resulting from deletion of each of the ORFs. Bioinformatic analysis revealed that YLL061w is probably an amino acid permease for S‐methylmethionine and that YLL060c encodes a glutathione transferase which is involved in cellular detoxification, while YLL058w may play a role in sulphur‐containing amino‐acid metabolism, YLL057c in sulphonate catabolism and YLL056c in stress response. The transcription of three ORFs (YLL061w, YLL057c and YLL056c) has been shown to increase more than 10‐fold under sulphate starvation. Replacement cassettes, comprising the kanMX marker flanked by each ORFs promoter and terminator regions, were cloned into pUG7. All the cognate clones, were generated using direct PCR products amplified from genomic DNA or using gap‐repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright

Functional analysis of six novel ORFs on the left arm of Chromosome XII in Saccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control

Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been analysed using short‐flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell growth; yll035w spores arrested after two or three cell divisions, while the majority of yll034c spores stopped growth within two cell cycles after germination. Complementation of the yll035w deletion with its cognate clone, and a promoter‐substitution experiment, indicated that the promoter of YLL035w may lie within the adjacent ORF, YLL036c. Transcriptional analysis demonstrated that YLL035w is under cell‐cycle regulation. Bioinformatic analyses produced significant matches between YLL034c and mammalian valosin and many other ATPases. The standard EUROFAN growth tests failed to reveal obvious phenotypes resulting from deletion of any of the four non‐essential ORFs. Replacement cassettes, comprising the kanMX marker flanked by each ORF’s promoter and terminator regions, were cloned into pUG7. All the cognate clones, except for YLL040c, were generated using direct PCR products amplified from genomic DNA or using gap‐repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright

Functional analysis of eight open reading frames on chromosomes XII and XIV of Saccharomyces cerevisiae

Deletion, together with basic functional and bioinformatic analyses has been carried out on eight novel ORFs discovered during the sequencing of the Saccharomyces cerevisiae genome. Six ORFs (YLL049w, YLL051c, YLL052c, YLL053c, YLL054c and YLL055w) located on the left arm, and one (YLR130c) on the right arm, of chromosome XII, and an eighth ORF (YNL331c) on the left arm of the chromosome XIV, have been investigated. ORFs were deleted by the SFH–PCR gene‐replacement strategy. Basic functional analysis revealed no obvious phenotype for any of the eight ORFs. Bioinformatic analysis, however, revealed possible functions for seven of the ORFs on the basis of the amino acid sequence similarity of their predicted protein products to those of proteins with known functions. ORF YLL051c (FRE6) shows similarity to iron transport proteins, such as ferric reductase. YLL052c and YLL053c appear to be aquaporins. The product of YLL054c (Yll054p) is highly similar to the oleate‐specific transcriptional activator protein (Pip2p), which is involved in the peroxisomal induction pathway (pip). ORF YLL055w is similar to Dal5p, allantoate permease, and may play role in allantoin transport. YLR130c (ZRT2) is a low‐affinity zinc transporter protein. YNL331c is also named AAD14, which is induced by chemicals that induce oxidative stress by depleting the cell of glutathione. Copyright

H2O2 stress‐specific regulation of S. pombe MAPK Sty1 by mitochondrial protein phosphatase Ptc4

In fission yeast, the stress‐activated MAP kinase, Sty1, is activated via phosphorylation upon exposure to stress and orchestrates an appropriate response. Its activity is attenuated by either serine/threonine PP2C or tyrosine phosphatases. Here, we found that the PP2C phosphatase, Ptc4, plays an important role in inactivating Sty1 specifically upon oxidative stress. Sty1 activity remains high in a ptc4 deletion mutant upon H2O2 but not under other types of stress. Surprisingly, Ptc4 localizes to the mitochondria and is targeted there by an N‐terminal mitochondrial targeting sequence (MTS), which is cleaved upon import. A fraction of Sty1 also localizes to the mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of the Ptc4 MTS is greatly reduced specifically upon H2O2, resulting in the full‐length form of the phosphatase; this displays a stronger interaction with Sty1, thus suggesting a novel mechanism by which the negative regulation of MAPK signalling is controlled and providing an explanation for the oxidative stress‐specific nature of the regulation of Sty1 by Ptc4.