scholarly journals Kinetic Features of 3′-5′ Exonuclease Activity of Human AP-Endonuclease APE1

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2101 ◽  
Author(s):  
Alexandra Kuznetsova ◽  
Olga Fedorova ◽  
Nikita Kuznetsov
Keyword(s):  
Kinetic Mechanism ◽  
Primary Role ◽  
Exonuclease Activity ◽  
Abasic Site ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Short Oligonucleotide ◽  
Rate Limiting Step ◽  
Ap Sites ◽  
The Impact

Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5′ to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3′-5′ exonuclease activity, which presumably is responsible for cleaning up nonconventional 3′ ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3′-end nucleotide removal in the 3′-5′ exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5′ dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3′ end of the short oligonucleotide. The impact of the 3′-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3′-5′ exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3′ nucleotide removal by APE1 in the 3′-5′ exonuclease process is the release of the detached nucleotide from the enzyme’s active site.

scholarly journals Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA

2018 ◽  
Vol 20 (1) ◽  
pp. 69 ◽  
Author(s):  
Wei-Wei Wang ◽  
Huan Zhou ◽  
Juan-Juan Xie ◽  
Gang-Shun Yi ◽  
Jian-Hua He ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Exonuclease Activity ◽  
Endonuclease Activity ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Phosphodiester Bonds ◽  
Ap Sites ◽  
Alkane Chain ◽  
Ap Site

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5′ of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3′-exonuclease activity for removing 3′-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3′-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5′ to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5′-terminus. The 3′-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3′-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

scholarly journals Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs

2021 ◽  
Vol 22 (16) ◽  
pp. 8763
Author(s):  
Marina Jordano-Raya ◽  
Cristina Beltrán-Melero ◽  
M. Dolores Moreno-Recio ◽  
M. Isabel Martínez-Macías ◽  
Rafael R. Ariza ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Lesions ◽  
Abasic Site ◽  
Base Loss ◽  
Base Pairs ◽  
Ap Endonuclease ◽  
Abasic Sites ◽  
Ap Sites ◽  
Ap Lyase ◽  
Ap Site

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.

scholarly journals Deoxyribonuclease IV from rat liver chromatin and the excision of apurinic sites from depurinated DNA

1985 ◽  
Vol 225 (2) ◽  
pp. 535-542 ◽  
Author(s):  
G Grondal-Zocchi ◽  
W G Verly
Keyword(s):  
Rat Liver ◽  
Liver Cells ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Double Stranded Dna ◽  
Rat Liver Cells ◽  
Liver Chromatin ◽  
Ap Sites ◽  
Beta Elimination ◽  
Hydrolysis Of

Deoxyribonuclease IV, a 5′-3′ exonuclease degrading double-stranded DNA from intra-strand nicks, has been purified from the chromatin of rat liver cells. The enzyme, which has an Mr of 58000, excises the apurinic (AP) sites from a depurinated DNA nicked 5′ to these AP sites with the chromatin AP endonuclease. The excision is not the result of hydrolysis of the phosphodiester bond 3′ to the AP sites since the excision product does not behave as deoxyribose 5-phosphate but as its 2,3-unsaturated derivative. This result suggests that, to remove the AP sites from the DNA nicked by an AP endonuclease, the chromatin deoxyribonuclease IV rather acts as a catalyst of beta-elimination.

scholarly journals Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

2018 ◽  
Vol 115 (5) ◽  
pp. E916-E924 ◽  
Author(s):  
Casimiro Barbado ◽  
Dolores Córdoba-Cañero ◽  
Rafael R. Ariza ◽  
Teresa Roldán-Arjona
Keyword(s):  
Excision Repair ◽  
Biochemical Properties ◽  
Abasic Site ◽  
Single Nucleotide ◽  
Ap Endonuclease ◽  
Abasic Sites ◽  
Lyase Activity ◽  
Ap Sites ◽  
Base Excision ◽  
Ap Lyase

Abasic (apurinic/apyrimidinic, AP) sites in DNA arise from spontaneous base loss or by enzymatic removal during base excision repair. It is commonly accepted that both classes of AP site have analogous biochemical properties and are equivalent substrates for AP endonucleases and AP lyases, although the relative roles of these two types of enzymes are not well understood. We provide here genetic and biochemical evidence that, in Arabidopsis, AP sites generated by spontaneous loss of N7-methylguanine (N7-meG) are exclusively repaired through an AP endonuclease-independent pathway initiated by FPG, a bifunctional DNA glycosylase with AP lyase activity. Abasic site incision catalyzed by FPG generates a single-nucleotide gap with a 3′-phosphate terminus that is processed by the DNA 3′-phosphatase ZDP before repair is completed. We further show that the major AP endonuclease in Arabidopsis (ARP) incises AP sites generated by enzymatic N7-meG excision but, unexpectedly, not those resulting from spontaneous N7-meG loss. These findings, which reveal previously undetected differences between products of enzymatic and nonenzymatic base release, may shed light on the evolution and biological roles of AP endonucleases and AP lyases.

scholarly journals The Impact of Microclimate on the Reproductive Phenology of Female Populus tomentosa in a Micro-Scale Urban Green Space in Beijing

2021 ◽  
Vol 13 (6) ◽  
pp. 3518
Author(s):  
Xiaoyi Xing ◽  
Li Dong ◽  
Cecil Konijnendijk ◽  
Peiyao Hao ◽  
Shuxin Fan ◽  
...  
Keyword(s):  
Seed Dispersal ◽  
Spatial Variation ◽  
Air Temperature ◽  
Green Space ◽  
Populus Tomentosa ◽  
Early Spring ◽  
Reproductive Phenology ◽  
Primary Role ◽  
Micro Scale ◽  
The Impact

The spatial variation of poplars’ reproductive phenology in Beijing’s urban area has aggravated the threat of poplar fluff (cotton-like flying seeds) to public health. This research explored the impact of microclimate conditions on the reproductive phenology of female Populus tomentosa in Taoranting Park, a micro-scale green space in Beijing (range <1 km). The observed phenophases covered flowering, fruiting, and seed dispersal, and ENVI-MET was applied to simulate the effect of the microclimate on SGS (start day of the growing season). The results showed that a significant spatial variation in poplar reproductive phenology existed at the research site. The variation was significantly affected by the microclimate factors DMT (daily mean temperature) and DMH (daily mean heat transfer coefficient), with air temperature playing a primary role. Specifically, the phenology of flowering and fruiting phenophases (BBB, BF, FF, FS) was negatively correlated with DMT (−0.983 ≤ r ≤ −0.908, p <0.01) and positively correlated with DMH (0.769 ≤ r ≤ 0.864, p < 0.05). In contrast, DSD (duration of seed dispersal) showed a positive correlation with DMT (r = 0.946, p < 0.01) and a negative correlation with DMH (r = −0.922, p < 0.01). Based on the findings, the increase in air convection with lower air temperature and decrease in microclimate variation in green space can be an effective way to shorten the seed-flying duration to tackle poplar fluff pollution in Beijing’s early spring.

Cancer patients’ awareness about clinical research in the era of social media and “fake news”: Preliminary results from ELPIS Study.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e24121-e24121
Author(s):  
Celeste Cagnazzo ◽  
Veronica Franchina ◽  
Giuseppe Toscano ◽  
Franca Fagioli ◽  
Tindara Franchina ◽  
...  
Keyword(s):  
Informed Consent ◽  
Clinical Research ◽  
Cancer Patients ◽  
Experimental Therapy ◽  
Average Score ◽  
Trial Participation ◽  
Primary Role ◽  
Fake News ◽  
Patient Awareness ◽  
The Impact

e24121 Background: Barriers for low recruitment in clinical trials have been classified based on three main sources: physician, patient, system. A primary role is played by a low patient awareness, which often leads to a lack of confidence in science and a substantial inability to estimate the benefits deriving from trial participation, aggravated by the spread of fake news. A prospective observational study was conducted to investigate the views of cancer patients on aspects of clinical research, their expectations, the level of comprehensibility of informed consent and the impact of the fake news phenomenon. Methods: From January 2018, after Ethics Committees approval, the ELPIS study was initiated in 9 Italian Medical Oncology Units. After signing the informed consent, patients were asked to complete a questionnaire, consisting of a set of multiple choice and Likert-score questions. Results: As of January 2021, 115 patients were enrolled, with a balanced sex distribution and a prevalence of subjects older than 55-years (79.8%). Regarding the previous knowledge about clinical research, the average score was 3.9 (range 1-5). The vast majority of respondents (91.3%) had already started experimental therapy and many of them constantly used internet (65.2%) and social networks (34.8%). More than half (53.9%) stated the interview with the physician was sufficient for a full understanding of informed consent. In case of doubt, the majority seeked support in the clinician (39.1%) while very few (1.7%) relied on the web. The average score attributed to doctor-patient relationship was equal to 8.89 (range 1-10). Respondents were quite confident in their ability to independently search for information on their disease, discriminate fake news and identify reliable sites (average score 3.26, 3.27, 3.09 respectively, over a range of 1-5). The scores related to the presumed ability to understand the results of a clinical study and to actively collaborate to produce research were high (average score 4.72 and 4.39 over a range of 1-5). Conclusions: Preliminary data from our research show a good level of patient awareness and a fine ability to understand information, discerning real from fake news. Continuing and implementing the training initiatives of the population in the health sector will certainly contribute to further improvement, hopefully obtaining an even greater involvement of patients in the early phases of research.

scholarly journals Hybrid Data Protection Framework to Enhance A2O Functionality in Production Database Virtualization

2020 ◽  
Vol 8 (6) ◽  
pp. 5691-5697
Keyword(s):  
Data Storage ◽  
Performance Indicator ◽  
Development Trend ◽  
Similar Data ◽  
Primary Role ◽  
City Development ◽  
Big Data Applications ◽  
Tightly Coupled ◽  
Deep Integration ◽  
The Impact

Deluge of information flows in the unprecedented scenario of smart city development trend, hence prone to issues on stability, reliability and availability. Smart data storage resources are vulnerable to provide functionality Always Available Online (A2O) due to their inherent heavy dependence on System Down Time (SDT), Redundant Systems and Software Failure (RS2F) or whole/ multiple site failures. In the absence of Production Database Management Services (PDMS), duplicate deployment of similar data on disjoint but similar architecture provides a Tightly Coupled Ultimate System (TCUS), which assures A2O mutually exclusive services. In this paper, we investigated active Data Guard (aDG) and Data Guard (DG) role management or switchover for a real time transition performed for database at standby state to cope up both planned maintenance and accidental RS2F events. We expose our results for deep integration of aDGs with ODB in-terms of Fast Sync to align synchronously at an ease of zero of wait states for disk I/O and configurability to Null Data Loss (NDL). Over a large range of remote or standby databases NDL make it certain to zero failover. The impact of aDG Fast-Start Failover in the cloud proximity make sure guaranteed NDL in synchronously and near NDL protection asynchronously. Hence, avoids unusual overhead impeding disk I/O and eventually on a primary database. We observe the key performance indicator in failover does not restart the standby database for primary role resumption, but introduce cloud proximity as a new primary database and the process is performed without any intervention of manual migration. The reliability of aDG Redo is flexible across not only standby databases but also primary sites running different operating system over diverse hardware platforms. The Redo capability enables migration with minimal downtime for any transaction in the clouds, therefore adds an inevitable functionality to big data applications.

scholarly journals Substrate specificity of sheep liver sorbitol dehydrogenase

1998 ◽  
Vol 330 (1) ◽  
pp. 479-487 ◽  
Author(s):  
I. Rune LINDSTAD ◽  
Peter KÖLL ◽  
John S. McKINLEY-McKEE
Keyword(s):  
Substrate Specificity ◽  
Ternary Complex ◽  
Kinetic Mechanism ◽  
Sorbitol Dehydrogenase ◽  
Hydroxyl Group ◽  
Enzyme Active Site ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

Chemical and biochemical properties of abasic site adducts and 6-thioguanine DNA cross-links in DNA

2017 ◽  
Author(s):  
◽  
Calvin D. Lewis
Keyword(s):  
Excision Repair ◽  
Biochemical Properties ◽  
Macromolecular Complex ◽  
Abasic Site ◽  
Cross Link ◽  
Dna Lesion ◽  
Phosphate Backbone ◽  
Cross Links ◽  
Ap Sites ◽  
Cellular Dna

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] DNA is a macromolecular complex, composed of the nucleotides adenine, thymine, guanine and cytosine interconnected by a phosphate backbone, that contains the genetic code for living organisms and viruses. Spontaneous and enzymatic hydrolysis of the glycosidic bonds that hold the coding nucleobases to the 2-deoxyribose-phosphate backbone of DNA results in the production of abasic (Ap) sites. These lesions are abundant in cellular DNA, and cellular Ap-containing DNA is damaging and may lead to cellular destruction if left unrepaired. Thus, efficient cellular DNA repair mechanisms that repair Ap sites have evolved in DNA containing organisms. The studies in this report examine the interaction between small molecules or naturally occurring DNA residues with Ap sites in duplex DNA. Experiments provide evidence that hydralazine binds to and forms a stable DNA lesion in single- and double-stranded DNA. Also, the hydralazine-DNA lesion is found to be a poor substrate for mammalian base excision repair enzymes such as Ap endonuclease and 8-oxoguanine DNA glycosylase. In addition, these studies provide preliminary evidence that hydralazine may potentiate the cytotoxicity of temozolomide in U87 cells. The investigation of the formation of cross-links between canonical DNA residues deoxyadenosine (dA) and deoxyguanosine (dG) with Ap sites is also explored. These experiments suggest that sequence effects contribute majorly to the cross-link yield in both dA- and dG-Ap site cross-links, especially when comparing central versus terminal cross-link locations. Here, this manuscript provides novel studies involving the interaction between DNA analog 6-thioguanine and opposing DNA bases in duplex oligonucleotide DNA.

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.

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