Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair.

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

Resilience, Anxiety, Stress, and Substance Use Patterns During COVID-19 Pandemic in the Miami Adult Studies on HIV (MASH) Cohort.

We evaluated mental health and substance use during the COVID-19 pandemic in 196 participants from the Miami Adult Studies on HIV (MASH) Cohort. A survey was administered between July-August of 2020, including validated measures of resilience and anxiety, a scale to measure COVID-19-related worry, and self-reported substance use. Compared to HIV-uninfected participants (n = 80), those living with HIV (n = 116) reported fewer anxiety symptoms, less COVID-19-related worry, and higher resilience. Those with more anxiety symptoms and lower resilience engaged in more frequent alcohol consumption, binge drinking, and cocaine use. Alcohol misuse was more common among HIV-uninfected participants. Cocaine use was reported by 21% fewer participants during the pandemic compared with 7.3 ± 1.5 months earlier. Possibly due to their experiences with HIV, PLWH responded with higher resilience and reduced worry and anxiety to the adversities brought by the COVID-19 pandemic.

Role of Selenium in Viral Infections with a Major Focus on SARS-CoV-2.

Viral infections have afflicted human health and despite great advancements in scientific knowledge and technologies, continue to affect our society today. The current coronavirus (COVID-19) pandemic has put a spotlight on the need to review the evidence on the impact of nutritional strategies to maintain a healthy immune system, particularly in instances where there are limited therapeutic treatments. Selenium, an essential trace element in humans, has a long history of lowering the occurrence and severity of viral infections. Much of the benefits derived from selenium are due to its incorporation into selenocysteine, an important component of proteins known as selenoproteins. Viral infections are associated with an increase in reactive oxygen species and may result in oxidative stress. Studies suggest that selenium deficiency alters immune response and viral infection by increasing oxidative stress and the rate of mutations in the viral genome, leading to an increase in pathogenicity and damage to the host. This review examines viral infections, including the novel SARS-CoV-2, in the context of selenium, in order to inform potential nutritional strategies to maintain a healthy immune system.

Trinucleotide repeat instability via DNA base excision repair.

Trinucleotide repeat (TNR) instability is the cause of over 40 human neurodegenerative diseases and certain types of cancer. TNR instability can result from DNA replication, repair, recombination, and gene transcription. Emerging evidence indicates that DNA base damage and base excision repair (BER) play an active role in regulating somatic TNR instability. These processes may potentially modulate the onset and progression of TNR-related diseases, given that TNRs are hotspots of DNA base damage that are present in mammalian cells with a high frequency. In this review, we discuss the recent advances in our understanding of the molecular mechanisms underlying BER-mediated TNR instability. We initially discuss the roles of the BER pathway and locations of DNA base lesions in TNRs and their interplay with non-B form DNA structures in governing repeat instability. We then discuss how the coordinated activities of BER enzymes can modulate a balance between the removal and addition of TNRs to regulate somatic TNR instability. We further discuss how this balance can be disrupted by the crosstalk between BER and DNA mismatch repair (MMR) machinery resulting in TNR expansion. Finally, we suggest future directions regarding BER-mediated somatic TNR instability and its association with TNR disease prevention and treatment.

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities.

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase ß (pol ß) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol ß loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol ß than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.

Diastereomeric Recognition of 5',8-cyclo-2'-Deoxyadenosine Lesions by Human Poly(ADP-ribose) Polymerase 1 in a Biomimetic Model.

5',8-Cyclo-2'-deoxyadenosine (cdA), in the 5'R and 5'Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by the nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the two diastereomeric cdA lesions could induce specific PARP1 binding. Here, we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions of 5'S-cdA and 5'R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism, fluorescence spectroscopy, immunoblotting analysis, and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1 were characterized. Remarkably, PARP1 exhibits different affinities in binding to a double strand (ds) oligonucleotide, which incorporates cdA lesions in R and S diastereomeric form. In particular, PARP1 proved to bind oligonucleotides, including a 5'S-cdA, with a higher affinity constant for the 5'S lesion in a model of ds DNA than 5'R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion.

Fluorescent 5-Pyrimidine and 8-Purine Nucleosides Modified with an N-Unsubstituted 1,2,3-Triazol-4-yl Moiety.

The Cu(I)- or Ag(I)-catalyzed cycloaddition between 8-ethynyladenine or guanine nucleosides and TMSN3 gave 8-(1- H-1,2,3-triazol-4-yl) nucleosides in good yields. On the other hand, reactions of 5-ethynyluracil or cytosine nucleosides with TMSN3 led to the chemoselective formation of triazoles via Cu(I)-catalyzed cycloaddition or vinyl azides via Ag(I)-catalyzed hydroazidation. These nucleosides with a minimalistic triazolyl modification showed excellent fluorescent properties with 8-(1- H-1,2,3-triazol-4-yl)-2'-deoxyadenosine (8-TrzdA), exhibiting a quantum yield of 44%. The 8-TrzdA 5'-triphosphate was incorporated into duplex DNA containing a one-nucleotide gap by DNA polymerase ß.

Pyrimidine Nucleosides with a Reactive (ß-Chlorovinyl)sulfone or (ß-Keto)sulfone Group at the C5 Position, Their Reactions with Nucleophiles and Electrophiles, and Their Polymerase-Catalyzed Incorporation into DNA.

Transition-metal-catalyzed chlorosulfonylation of 5-ethynylpyrimidine nucleosides provided (E)-5-(ß-chlorovinyl)sulfones A, which undergo nucleophilic substitution with amines or thiols affording B. The treatment of vinyl sulfones A with ammonia followed by acid-catalyzed hydrolysis of the intermediary ß-sulfonylvinylamines gave 5-(ß-keto)sulfones C. The latter reacts with electrophiles, yielding α-carbon-alkylated or -sulfanylated analogues D. The 5'-triphosphates of A and C were incorporated into double-stranded DNA, using open and one-nucleotide gap substrates, by human or Escherichia coli DNA-polymerase-catalyzed reactions.

Modulation of trinucleotide repeat instability by DNA polymerase ß polymorphic variant R137Q.

Trinucleotide repeat (TNR) instability is associated with human neurodegenerative diseases and cancer. Recent studies have pointed out that DNA base excision repair (BER) mediated by DNA polymerase ß (pol ß) plays a crucial role in governing somatic TNR instability in a damage-location dependent manner. It has been shown that the activities and function of BER enzymes and cofactors can be modulated by their polymorphic variations. This could alter the function of BER in regulating TNR instability. However, the roles of BER polymorphism in modulating TNR instability remain to be elucidated. A previous study has shown that a pol ß polymorphic variant, polßR137Q is associated with cancer due to its impaired polymerase activity and its deficiency in interacting with a BER cofactor, proliferating cell nuclear antigen (PCNA). In this study, we have studied the effect of the pol ßR137Q variant on TNR instability. We showed that pol ßR137Q exhibited weak DNA synthesis activity to cause TNR deletion during BER. We demonstrated that similar to wild-type pol ß, the weak DNA synthesis activity of pol ßR137Q allowed it to skip over a small loop formed on the template strand, thereby facilitating TNR deletion during BER. Our results further suggest that carriers with pol ßR137Q polymorphic variant may not exhibit an elevated risk of developing human diseases that are associated with TNR instability.

Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase ß during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase ß (pol ß) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol ß wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol ß knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol ß plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol ß inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol ß, indicating a mutagenic effect induced by pol ß lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol ß generated an intermediate that failed to be extended by pol ß, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol ß plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol ß bypassing of a cdA lesion.

AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair.

Base excision repair (BER) of an oxidized base within a trinucleotide repeat (TNR) tract can lead to TNR expansions that are associated with over 40 human neurodegenerative diseases. This occurs as a result of DNA secondary structures such as hairpins formed during repair. We have previously shown that BER in a TNR hairpin loop can lead to removal of the hairpin, attenuating or preventing TNR expansions. Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3'-5' exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop. Coordinating with flap endonuclease 1, the APE1 3'-5' exonuclease activity cleaves the annealed upstream 3'-flap of a double-flap intermediate resulting from 5'-incision of an abasic site in the hairpin loop. Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions.

Mortalidad perinatal y duelo materno / Perinatal mortality and maternal sorrow

Se investigaron desde el punto de vista médico-psicológico dos grupos de madres cuyos hijos murieron durante el período perinatal (entre 28 semanas de gestación y 28 días de nacido). El primer grupo de estudio lo configuraban 20 pacientes que reaccionaron con un duelo prolongado ante la pérdida de su hijo. El segundo grupo está conformado por 20 pacientes (grupo de comparación) que elaboraron la pérdida de su hijo mediante un duelo no complicado. La hipótesis general que orientó este trabajo es la siguiente: El vínculo establecido entre la madre y su hijo real, en caso de que éste muera, facilita la elaboración del duelo; por otra parte la insuficiencia o carencia del vínculo con el hijo real, aumenta la relación de la madre con su hijo imaginario de tal manera que si el niño muere la relación interna madre-bebé de tipo narcisista tiende a complicar el duelo prolongándolo y distorsionándolo. "Hijo real" se refiere al hijo como objeto externo e "hijo imaginario", es la representación vivencial del hijo u objeto interno. Esta hipótesis general se subdividió en cinco hipótesis simples, con fines operativos. Los resultados confirmaron la hipótesis básica, al establecer que las variables indicadoras o facilitadoras del vínculo mostraron una diferencia significativa desde el punto de vista estadístico entre los dos grupos. Estas variables son: contacto visual, contacto táctil entre la madre y el hijo, información de la condición del bebé, participación activa de la madre en la asignación de un nombre para su hijo y en la asistencia a las ceremonias funerarias. Se hacen algunas recomendaciones prácticas para el personal de los servicios de maternidad y perinatología en el sentido de propiciar y facilitar el contacto entre la madre y su hijo lo cual a su vez activa el establecimiento de un vínculo emocional y la elaboración de la pérdida del bebé en caso de que éste fallezca

Embarazo en adolescentes: repercusiones psíquicas

Este trabajo clínico estableció como objetivos principales identificar el impacto psicológico que tiene sobre madres solteras adolescentes tempranas un embarazo no deseado, el tipo de trastorno psiquiátrico que puedan presentar y cómo se altera el vínculo con su bebé. El diseño metodológico comprendió la estructuración de dos grupos de pacientes: de estudio y de comparación. En ambos grupos se estudiaron las características demográficas, ambiente familiar, situación de pareja, algunas de las condiciones bajo las cuales se adelantó el embarazo, así como el destino de la relación madre-bebé y las principales complicaciones médicas y psiquiátricas. Se analizaron las diferencias estadísticamente significativas encontradas, demostrando la existencia de una mayor vulnerabilidad psíquica de las adolescentes tempranas, acompañada de la presencia de un mayor número de complicaciones obstétricas y de una falla de la capacidad de asumir el rol de madres. Se revisó la bibliografía médica pertinente al tema entre los años 1970-1990, cubriendo los siguientes tópicos: epidemiología, cambios psíquicos durante el embarazo, complicaciones obstétricas, consecuencias socioeconómicas y aspectos legales. Se encontraron vacíos de información en lo relacionado con: estadística acerca del devenir de estos embarazos, principalmente en lo relacionado con el aborto (en países en vía de desarrollo) y la adopción (a nivel general), cuantificación de costos generados por la problemática del embarazo en la adolescencia en nuestro país, la legislación en cuanto a la educación sexual y los derechos del adolescente.