Single Stranded Dna
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2021 ◽  
Author(s):  
Tomoko Tanaka ◽  
Shinobu Hirai ◽  
Hiroyuki Manabe ◽  
Kentaro Endo ◽  
Hiroko Shimbo ◽  
...  

Aging involves a decline in physiology which is a natural event in all living organisms. An accumulation of DNA damage contributes to the progression of aging. DNA is continually damaged by exogenous sources and endogenous sources. If the DNA repair pathway operates normally, DNA damage is not life threatening. However, impairments of the DNA repair pathway may result in an accumulation of DNA damage, which has a harmful effect on health and causes an onset of pathology. RP58, a zinc-finger transcriptional repressor, plays a critical role in cerebral cortex formation. Recently, it has been reported that the expression level of RP58 decreases in the aged human cortex. Furthermore, the role of RP58 in DNA damage is inferred by the involvement of DNMT3, which acts as a co-repressor for RP58, in DNA damage. Therefore, RP58 may play a crucial role in the DNA damage associated with aging. In the present study, we investigated the role of RP58 in aging. We used RP58 hetero-knockout and wild-type mice in adolescence, adulthood, or old age. We performed immunohistochemistry to determine whether microglia and DNA damage markers responded to the decline in RP58 levels. Furthermore, we performed an object location test to measure cognitive function, which decline with age. We found that the wild-type mice showed an increase in single-stranded DNA and gamma-H2AX foci. These results indicate an increase in DNA damage or dysfunction of DNA repair mechanisms in the hippocampus as age-related changes. Furthermore, we found that, with advancing age, both the wild-type and hetero-knockout mice showed an impairment of spatial memory for the object and increase in reactive microglia in the hippocampus. However, the RP58 hetero-knockout mice showed these symptoms earlier than the wild-type mice did. These results suggest that a decline in RP58 level may lead to the progression of aging.


2021 ◽  
Vol 104 (3) ◽  
Author(s):  
Qiujin Wang ◽  
Shuo Lin ◽  
Xuan Liu ◽  
Wen Xu ◽  
Yiming Xiao ◽  
...  

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mário Špírek ◽  
Martin R. G. Taylor ◽  
Ondrej Belan ◽  
Simon J. Boulton ◽  
Lumir Krejci

AbstractThe RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that ‘nucleotide proofreading’ activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abid Javed ◽  
Balazs Major ◽  
Jonathan A. Stead ◽  
Cyril M. Sanders ◽  
Elena V. Orlova

AbstractHexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged β-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.


2021 ◽  
Author(s):  
Alina Tarsalainen ◽  
Yaakov Maman ◽  
Fei-Long Meng ◽  
Minna K. Kylaniemi ◽  
Anni Soikkeli ◽  
...  

Somatic hypermutation (SHM) drives the genetic diversity of immunoglobulin (Ig) genes in activated B cells and supports the generation of antibodies with increased affinity for antigen. SHM is targeted to Ig genes by their enhancers (DIVACs; diversification activators), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase 2 (Pol2) and Pol2 occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol2 or production of full-length transcripts, indicating accumulation of stalled Pol2. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of single-stranded DNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol2 stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.


2021 ◽  
Vol 118 (38) ◽  
pp. e2109334118
Author(s):  
Albert Serra-Cardona ◽  
Chuanhe Yu ◽  
Xinmin Zhang ◽  
Xu Hua ◽  
Yuan Yao ◽  
...  

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.


2021 ◽  
Author(s):  
Liang Cai ◽  
Krishna M Kovur ◽  
Prashanthi Kovur ◽  
Carlo D. Montemagno

We report the design, fabrication and quantitative performance analysis of a low-cost, flexible carbon nanotube (CNT) network-based deoxyribonucleic acid (DNA) sensor. These sensors comprise an array of ink-jet printed silver (Ag) electrodes on a transparent polyethylene terephthalate (PET) flexible substrate, where a CNT network acts as a sensing layer. The DNA hybridization is studied by immobilizing single-stranded DNA (ssDNA) probes on the CNT surface; these probes recognize their complementary DNA target. Further, we have carried out a quantitative performance analysis of the flexible CNT biosensors using the analytic hierarchy process (AHP). We have identified various influencing factors and sub-factors (performance indicators), and quantified the performance of the flexible CNT biosensors in different measured states (before bending, during bending and after bending). Additionally, the noise and other external factors contributing to the measured real signal have been quantified. The interpretation of the overall outcome will enable the improvement of the performance of flexible biosensors fabricated through large-scale manufacturing for possible commercialization.


2021 ◽  
Author(s):  
Liang Cai ◽  
Krishna M Kovur ◽  
Prashanthi Kovur ◽  
Carlo D. Montemagno

We report the design, fabrication and quantitative performance analysis of a low-cost, flexible carbon nanotube (CNT) network-based deoxyribonucleic acid (DNA) sensor. These sensors comprise an array of ink-jet printed silver (Ag) electrodes on a transparent polyethylene terephthalate (PET) flexible substrate, where a CNT network acts as a sensing layer. The DNA hybridization is studied by immobilizing single-stranded DNA (ssDNA) probes on the CNT surface; these probes recognize their complementary DNA target. Further, we have carried out a quantitative performance analysis of the flexible CNT biosensors using the analytic hierarchy process (AHP). We have identified various influencing factors and sub-factors (performance indicators), and quantified the performance of the flexible CNT biosensors in different measured states (before bending, during bending and after bending). Additionally, the noise and other external factors contributing to the measured real signal have been quantified. The interpretation of the overall outcome will enable the improvement of the performance of flexible biosensors fabricated through large-scale manufacturing for possible commercialization.


PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009807
Author(s):  
Marco Gnugnoli ◽  
Erika Casari ◽  
Maria Pia Longhese

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nuclease Exo1 or Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.


2021 ◽  
Vol 12 ◽  
Author(s):  
Luoshan Ruan ◽  
Xin Li

Nucleic acid aptamers are short single-stranded DNA or RNA oligonucleotides selected from a random single-stranded nucleic acid library using systematic evolution of ligands by exponential enrichment technology. To allow them to bind to molecular targets with the same specificity and precision as that of antibodies, aptamers are folded into secondary or tertiary structures. However, compared to antibodies, aptamers are not immunogenic and are easier to synthesize. Furthermore, they are chemically modified, which protects them from degradation by nucleases. Hence, due to their stability and favorable targeting ability, aptamers are promising for the diagnosis and treatment of diseases. Ovarian cancer has the worst prognosis among all gynecological diseases and is usually diagnosed at the medium and advanced stages due to its nonspecific symptoms. Relapse is common, even if patients receive a standard therapeutic regimen including surgery and chemotherapy; simultaneously, drug resistance and adverse effects are reported in a several patients. Therefore, the safer and more efficient diagnostic and treatment method for ovarian cancer is imperative. Scientists have been trying to utilize aptamer technology for the early diagnosis and accurate treatment of ovarian cancer and some progress has been made in this field. This review discusses the screening of nucleic acid aptamers by targeting ovarian cancer cells and the application of aptamers in the diagnosis and treatment of ovarian cancer.


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