Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

Otsuka, Chie; Kobayashi, Keita; Kawaguchi, Naho; Kunitomi, Nozomu; Moriyama, K.; Hata, Yoshihiro; Iwai, Shigenori; Loakes, David; Noskov, Vladimir N.; Pavlov, Youri I.; Negishi, Kazuo

doi:10.1016/s0027-5107(02)00023-4

Cited by 21 publications

(17 citation statements)

References 25 publications

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“…Single-stranded DNA oligos can be used to produce small genetic modifications via a simple transformation procedure (69). Because the transformation efficiency may be reduced by the endogenous mismatch repair system (70), MSH2 (YOL090W), a DNA mismatch repair gene, was inactivated by partial deletion and replaced by a kanamycin-resistance marker (KanMX4) (Fig.…”

Section: Inactivation Of Msh2 (Muts Homolog 2)mentioning

confidence: 99%

Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast

Dvir

Velten

Sharon

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

240

287

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The 5′-untranslated region (5′-UTR) of mRNAs contains elements that affect expression, yet the rules by which these regions exert their effect are poorly understood. Here, we studied the impact of 5′-UTR sequences on protein levels in yeast, by constructing a large-scale library of mutants that differ only in the 10 bp preceding the translational start site of a fluorescent reporter. Using a high-throughput sequencing strategy, we obtained highly accurate measurements of protein abundance for over 2,000 unique sequence variants. The resulting pool spanned an approximately sevenfold range of protein levels, demonstrating the powerful consequences of sequence manipulations of even 1-10 nucleotides immediately upstream of the start codon. We devised computational models that predicted over 70% of the measured expression variability in held-out sequence variants. Notably, a combined model of the most prominent features successfully explained protein abundance in an additional, independently constructed library, whose nucleotide composition differed greatly from the library used to parameterize the model. Our analysis reveals the dominant contribution of the start codon context at positions −3 to −1, mRNA secondary structure, and out-of-frame upstream AUGs (uAUGs) to phenotypic diversity, thereby advancing our understanding of how protein levels are modulated by 5′-UTR sequences, and paving the way toward predictably tuning protein expression through manipulations of 5′-UTRs.post-transcriptional regulation | computational prediction | AUG sequence context | mRNA folding | upstream start codons T he control of protein expression is a fundamental process of living cells. Much progress has recently been made in understanding how information encoded within DNA regulatory sequences is converted to yield a precise expression output (1-4). However, functional elements are also embedded within RNA sequences, such as 5′-untranslated regions (5′-UTRs). Accumulating evidence has revealed the importance of 5′-UTRs in shaping eukaryotic protein expression (5-12). Such 5′-UTR sequences encode a variety of cis-regulatory elements, including a 5′-cap structure (13), a translation initiation motif (14-16), upstream AUGs (uAUGs) and upstream ORFs (17, 18), internal ribosome entry sites (19), terminal oligo-pyrimidine tracts (20), secondary structures (21), and G-quadruplexes (22). Many of these elements were shown to control protein levels by altering the efficiency of translation (7-9, 11), whereas some elements affect translation and in addition, transcription (15) or mRNA degradation (23). Despite much progress, significant challenges remain in deciphering the rules by which multiple elements combine to fine-tune protein expression, as well as in quantifying the degree of expression variation that may be jointly explained by known and novel regulatory elements.Traditionally, the impact of 5′-UTR elements on protein abundance is determined experimentally by comparing a perturbed 5′-UTR sequence with an appropriate control (16,(23)(2...

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Section: Inactivation Of Msh2 (Muts Homolog 2)mentioning

confidence: 99%

Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast

Dvir

Velten

Sharon

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

240

287

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“…Specifically, the use of ssOligos with differing sensitivities to MutS␣ and MutS␤ has revealed striking and unexpected strand differences in the activity of the two MMR complexes as well as unexpected differences in response to loss of MLH1. With a better understanding of the mechanism in place, this method should prove extremely valuable in studying the effects of defined base mismatches and damage on repair and replication in a chromosomal context (44)(45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

“…Yeast transformation was performed by electroporation following the general procedure of Otsuka et al (46). Briefly, an overnight culture of yeast cells (25 ml) was inoculated into 900 ml of YPDA medium (yeast-peptone-dextrose containing 0.0075% l-adenine hemisulfate) and cultured until the OD 600 reached 1.3-1.5.…”

Section: Methodsmentioning

confidence: 99%

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

Kow¹,

Bao²,

Reeves

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Transformation of both prokaryotes and eukaryotes with singlestranded oligonucleotides can transfer sequence information from the oligonucleotide to the chromosome. We have studied this process using oligonucleotides that correct a ؊1 frameshift mutation in the LYS2 gene of Saccharomyces cerevisiae. We demonstrate that transformation by oligonucleotides occurs preferentially on the lagging strand of replication and is strongly inhibited by the mismatch-repair system. These results are consistent with a mechanism in which oligonucleotides anneal to single-stranded regions of DNA at a replication fork and serve as primers for DNA synthesis. Because the mispairs the primers create are efficiently removed by the mismatch-repair system, single-stranded oligonucleotides can be used to probe mismatch-repair function in a chromosomal context. Removal of mispairs created by annealing of the single-stranded oligonucleotides to the chromosomal DNA is as expected, with 7-nt loops being recognized solely by MutS␤ and 1-nt loops being recognized by both MutS␣ and MutS␤. We also find evidence for Mlh1-independent repair of 7-nt, but not 1-nt, loops. Unexpectedly, we find a strand asymmetry of mismatchrepair function; transformation is blocked more efficiently by MutS␣ on the lagging strand of replication, whereas MutS␤ does not show a significant strand bias. These results suggest an inherent strand-related difference in how the yeast MutS␣ and MutS␤ complexes access and/or repair mismatches that arise in the context of DNA replication.T he transformation of yeast by single-stranded oligonucleotides (ssOligos) was first demonstrated 20 years ago by Sherman and colleagues (1). By using the CYC1 gene as a target of ssOligos of 50 nt, it was found that mismatches, in general, reduced ssOligo transformation (ssOT) efficiency, that 3Ј deoxyoligonucleotides transformed somewhat less well than those with a 3Ј hydroxyl and that ssOT was independent of tested recombination functions (2, 3). A strong strand bias also was observed, with ssOligos having the sequence of the coding strand of CYC1 (defined here as COD oligonucleotides) transforming 50-100 times better than oligonucleotides with the complementary sequence (noncoding, or NC, oligonucleotides). It was suggested that the differences between COD and NC ssOT frequencies were not related to transcriptional differences but could be due to preferential incorporation of oligonucleotides into either the leading or lagging strand of replication (3). More recently, the Kmiec (4) lab has studied the transformation of cyc1 point mutants using 70-nt ssOligos with three phosphorothioate bonds at the 3Ј and 5Ј termini. In this case, there appeared to be an opposite strand bias, with NC ssOligos transforming better than COD ssOligos. Furthermore, it was suggested that this transformation was enhanced by repair processes such as mismatch repair (MMR), with transformation apparently decreasing in MMR-defective cells (4).In addition to the yeast studies, it has also been shown that ssOligos can t...

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“…Lesions have been engineered into gapped plasmids (Nelson et al 2000;Gibbs et al 2005), double-strand plasmids (Baynton et al 1998(Baynton et al , 1999Pagès et al 2008a,b), and single-strand plasmids (Zhao et al 2006(Zhao et al , 2010. Additionally, a single-strand oligonucleotide containing a defined lesion can be introduced into the genome by transformation, where it serves as a template in the subsequent round of DNA replication (Otsuka et al 2002a(Otsuka et al ,b, 2005Kow et al 2005;Bao and Kow 2009). Finally, in vitro assays with purified proteins afford the opportunity to assess the individual steps of TLS through manipulating primer-template combinations and/or the nucleotide pool.…”

Section: Components Of Error-free and Error-prone Tlsmentioning

confidence: 99%

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

2013

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DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage.

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Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

Cited by 21 publications

References 25 publications

Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast

Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

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