Large deletions and untargeted substitutions induced by abasic site analog on leading versus lagging strand templates in human cells

Mutagenesis ◽  
2019 ◽  
Author(s):  
Tetsuya Suzuki ◽  
Yuri Katayama ◽  
Yasuo Komatsu ◽  
Hiroyuki Kamiya
Keyword(s):  
Large Deletion ◽  
Indicator Strain ◽  
Human Cells ◽  
Abasic Site ◽  
Abasic Sites ◽  
Large Deletions ◽  
Specific Position ◽  
Leading Strand ◽  
Supf Gene ◽  
Lagging Strand

Abstract The tetrahydrofuran-type abasic site analog (THF) induces large deletion mutations in human cells. To compare the large deletions induced by THF on leading and lagging strand templates, plasmid DNAs bearing the analog at a specific position outside the supF gene were introduced into human U2OS cells. The replicated DNAs recovered from the transfected cells were electroporated into an Escherichia coli indicator strain. THF on the lagging strand template produced more supF mutants than THF on the leading strand template. This unequal mutagenicity was due to the higher frequencies of not only large deletions but also untargeted base substitutions induced in the gene. These results suggested that both types of mutations occur more frequently when abasic sites are formed on the lagging strand template.

scholarly journals Analysis of large deletion mutations induced by abasic site analog in human cells

2018 ◽  
Vol 40 (1) ◽  
Author(s):  
Tetsuya Suzuki ◽  
Yuri Katayama ◽  
Yasuo Komatsu ◽  
Hiroyuki Kamiya
Keyword(s):  
Large Deletion ◽  
Human Cells ◽  
Abasic Site ◽  
Deletion Mutations

scholarly journals Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

2016 ◽  
Vol 2016 ◽  
pp. 1-4
Author(s):  
Chun Wu
Keyword(s):  
Dna Polymerase ◽  
Click Reaction ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sequencing Analysis ◽  
Abasic Site ◽  
Nylon Membrane ◽  
Abasic Sites ◽  
Polymerase Chain ◽  
Dna Sequencing Analysis ◽  
Endonuclease Digestion

Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.

scholarly journals High-resolution EPR distance measurements on RNA and DNA with the non-covalent Ǵ spin label

2019 ◽  
Vol 48 (2) ◽  
pp. 924-933 ◽  
Author(s):  
Marcel Heinz ◽  
Nicole Erlenbach ◽  
Lukas S Stelzl ◽  
Grace Thierolf ◽  
Nilesh R Kamble ◽  
...  
Keyword(s):  
High Resolution ◽  
Nucleic Acids ◽  
Spin Label ◽  
Double Resonance ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Spin Labels ◽  
Abasic Site ◽  
Abasic Sites ◽  
Rna And Dna

Abstract Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

scholarly journals Molecular basis of abasic site sensing in single-stranded DNA by the SRAP domain of E. coli yedK

2019 ◽  
Vol 47 (19) ◽  
pp. 10388-10399 ◽  
Author(s):  
Na Wang ◽  
Hongyu Bao ◽  
Liu Chen ◽  
Yanhong Liu ◽  
Yue Li ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Substrate Binding ◽  
Specific Substrate ◽  
Abasic Site ◽  
E Coli ◽  
Abasic Sites ◽  
Ap Sites ◽  
Ap Site

Abstract HMCES and yedK were recently identified as sensors of abasic sites in ssDNA. In this study, we present multiple crystal structures captured in the apo-, nonspecific-substrate-binding, specific-substrate-binding, and product-binding states of yedK. In combination with biochemical data, we unveil the molecular basis of AP site sensing in ssDNA by yedK. Our results indicate that yedK has a strong preference for AP site-containing ssDNA over native ssDNA and that the conserved Glu105 residue is important for identifying AP sites in ssDNA. Moreover, our results reveal that a thiazolidine linkage is formed between yedK and AP sites in ssDNA, with the residues that stabilize the thiazolidine linkage important for the formation of DNA-protein crosslinks between yedK and the AP sites. We propose that our findings offer a unique platform to develop yedK and other SRAP domain-containing proteins as tools for detecting abasic sites in vitro and in vivo.

Effects of the C4‘-Oxidized Abasic Site on Replication inEscherichia coli. An Unusually Large Deletion Is Induced by a Small Lesion†

Biochemistry ◽  
2004 ◽  
Vol 43 (43) ◽  
pp. 13621-13627 ◽  
Author(s):  
Kelly M. Kroeger ◽  
Jaeseung Kim ◽  
Myron F. Goodman ◽  
Marc M. Greenberg
Keyword(s):  
Large Deletion ◽  
Small Lesion ◽  
Inescherichia Coli ◽  
Abasic Site ◽  
Oxidized Abasic Site

Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia

Blood ◽  
2012 ◽  
Vol 119 (10) ◽  
pp. 2376-2384 ◽  
Author(s):  
Madoka Kuramitsu ◽  
Aiko Sato-Otsubo ◽  
Tomohiro Morio ◽  
Masatoshi Takagi ◽  
Tsutomu Toki ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Single Nucleotide Polymorphism Array ◽  
Gene Copy Number ◽  
Japanese Patients ◽  
Large Deletion ◽  
Gene Copy ◽  
Easy Method ◽  
Diamond Blackfan Anemia ◽  
Large Deletions ◽  
Novel Approach

Abstract Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

scholarly journals Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II

2018 ◽  
Vol 115 (11) ◽  
pp. E2538-E2545 ◽  
Author(s):  
Wei Wang ◽  
Celine Walmacq ◽  
Jenny Chong ◽  
Mikhail Kashlev ◽  
Dong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Lesions ◽  
Structural Motif ◽  
Structural Basis ◽  
Dependent Manner ◽  
Abasic Site ◽  
Structural Explanation ◽  
Pol Ii ◽  
Abasic Sites

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3′-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

scholarly journals The Effects of Molecular Crowding on the Structure and Stability of G-Quadruplexes with an Abasic Site

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Takeshi Fujimoto ◽  
Shu-ichi Nakano ◽  
Daisuke Miyoshi ◽  
Naoki Sugimoto
Keyword(s):  
Site Effects ◽  
Enthalpy Change ◽  
Molecular Crowding ◽  
Abasic Site ◽  
Structure And Stability ◽  
Quadruplex Structure ◽  
Abasic Sites ◽  
Crowding Effects ◽  
G Quadruplex ◽  
Quadruplex Formation

Both cellular environmental factors and chemical modifications critically affect the properties of nucleic acids. However, the structure and stability of DNA containing abasic sites under cell-mimicking molecular crowding conditions remain unclear. Here, we investigated the molecular crowding effects on the structure and stability of the G-quadruplexes including a single abasic site. Structural analysis by circular dichroism showed that molecular crowding by PEG200 did not affect the topology of the G-quadruplex structure with or without an abasic site. Thermodynamic analysis further demonstrated that the degree of stabilization of the G-quadruplex by molecular crowding decreased with substitution of an abasic site for a single guanine. Notably, we found that the molecular crowding effects on the enthalpy change for G-quadruplex formation had a linear relationship with the abasic site effects depending on its position. These results are useful for predicting the structure and stability of G-quadruplexes with abasic sites in the cell-mimicking conditions.

scholarly journals Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

2019 ◽  
Vol 116 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Glen E. Cronan ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Chromosomal Dna ◽  
E Coli ◽  
Okazaki Fragments ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

scholarly journals Replicative Bypass of Abasic Site in Escherichia coli and Human Cells: Similarities and Differences

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e107915 ◽  
Author(s):  
Savithri Weerasooriya ◽  
Vijay P. Jasti ◽  
Ashis K. Basu
Keyword(s):  
Escherichia Coli ◽  
Human Cells ◽  
Abasic Site ◽  
Similarities And Differences
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