Engineering Erg10 Thiolase from Saccharomyces cerevisiae as a Synthetic Toolkit for the Production of Branched-Chain Alcohols.

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.

Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage.

We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T(722)A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways.

Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the Rad1-Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1-Rad10 endonuclease cleaves 3' branches of DNA and aberrant 3' DNA ends that are refractory to other 3' processing enzymes. Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB sites depends on both RAD51 and RAD52, but not MRE11 while IR-induced breaks do not require RAD51. Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1-Rad10 complex in DSB repair in yeast.

More is not always better: the genetic constraints of polyploidy.

Polyploid cells are a characteristic feature of certain human tissues, and notably many cancers. In a systematic genomic screen in yeast, Storchová and co-workers identified the genetic requirements of tetraploidy. Surprisingly, they showed that only three connected pathways are essential for the viability of tetraploid yeast cells. These data provide exciting new targets that might be essential specifically in polyploid cancer cells.

Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe.

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3'-->5' proofreading activity and its preferred substrates are small gaps with a 5'-phosphate group. Similarly to Polmu, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1-2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polmu, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polmu. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polbeta and Pollambda, and unlike Polmu, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polbeta, Pollambda and Polmu.

Defective nucleotide excision repair in yeast hpr1 and tho2 mutants.

Nucleotide excision repair (NER) and transcription are intimately related. First, TFIIH has a dual role in transcription initiation and NER and, secondly, transcription leads to more efficient repair of damage present in transcribed sequences. It is thought that elongating RNAPII, stalled at a DNA lesion, is used for the loading of the NER machinery in a process termed transcription-coupled repair (TCR). Non-transcribed regions are repaired by the so-called global genome repair (GGR). We have previously defined a number of yeast genes, whose deletions confer transcription-dependent hyper-recombination phenotypes. As these mutations cause impairment of transcription elongation we have assayed whether they also affect DNA repair. We show that null mutations of the HPR1 and THO2 genes, encoding two prominent proteins of the THO complex, increase UV sensitivity of yeast cells lacking GGR. Consistent with this result, molecular analyses of DNA repair of the RPB2 transcribed strand using T4 endo V show that hpr1 and tho2 do indeed impair TCR. However, this effect is not confined to TCR alone because the mutants are slightly affected in GGR. These results indicate that THO affects both transcription and NER. We discuss different alternatives to explain the effect of the THO complex on DNA repair.

Lack of sugar discrimination by human Pol mu requires a single glycine residue.

DNA polymerase mu (Pol mu) is a novel family X DNA polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol mu is not able to discriminate against the 2'-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol mu is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol mu based on the available Pol beta and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol mu was mutated to the consensus tyrosine present in Pol beta, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol mu in DNA repair.

Transcription and double-strand breaks induce similar mitotic recombination events in Saccharomyces cerevisiae.

We have made a comparative analysis of double-strand-break (DSB)-induced recombination and spontaneous recombination under low- and high-transcription conditions in yeast. We constructed two different recombination substrates, one for the analysis of intermolecular gene conversions and the other for intramolecular gene conversions and inversions. Such substrates were based on the same leu2-HOr allele fused to the tet promoter and containing a 21-bp HO site. Gene conversions and inversions were differently affected by rad1, rad51, rad52, and rad59 single and double mutations, consistent with the actual view that such events occur by different recombination mechanisms. However, the effect of each mutation on each type of recombination event was the same, whether associated with transcription or induced by the HO-mediated DSB. Both the highly transcribed DNA and the HO-cut sequence acted as recipients of the gene conversion events. These results are consistent with the hypothesis that transcription promotes initiation of recombination along the DNA sequence being transcribed. The similarity between transcription-associated and DSB-induced recombination suggests that transcription promotes DNA breaks.

Molecular evidence for a positive role of Spt4 in transcription elongation.

We have previously shown that yeast mutants of the THO complex have a defect in gene expression, observed as an impairment of lacZ transcription. Here we analyze the ability of mutants of different transcription elongation factors to transcribe lacZ. We found that spt4Delta, like THO mutants, impaired transcription of lacZ and of long and GC-rich DNA sequences fused to the GAL1 promoter. Using a newly developed in vitro transcription elongation assay, we show that Spt4 is required in elongation. There is a functional interaction between Spt4 and THO, detected by the lethality or strong gene expression defect and hyper-recombination phenotypes of double mutants in the W303 genetic background. Our results indicate that Spt4-Spt5 has a positive role in transcription elongation and suggest that Spt4-Spt5 and THO act at different steps during mRNA biogenesis.

Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast.

Equal sister chromatid exchange (SCE) has been thought to be an important mechanism of double-strand break (DSB) repair in eukaryotes, but this has never been proven due to the difficulty of distinguishing SCE products from parental molecules. To evaluate the biological relevance of equal SCE in DSB repair and to understand the underlying molecular mechanism, we developed recombination substrates for the analysis of DSB repair by SCE in yeast. In these substrates, most breaks are limited to one chromatid, allowing the intact sister chromatid to serve as the repair template; both equal and unequal SCE can be detected. We show that equal SCE is a major mechanism of DSB repair, is Rad51 dependent, and is stimulated by Rad59 and Mre11. Our work provides a physical analysis of mitotically occurring SCE in vivo and opens new perspectives for the study and understanding of DSB repair in eukaryotes.