A common transcriptional mechanism involving R-loop and RNA abasic site regulates an enhancer RNA of APOE.

RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II.

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated.

RNA abasic sites in yeast and human cells.

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group.

Histone H3 Lysine 56 Acetylation Enhances AP Endonuclease 1-Mediated Repair of AP Sites in Nucleosome Core Particles.

Deciphering factors modulating DNA repair in chromatin is of great interest because nucleosomal positioning influences mutation rates. H3K56 acetylation (Ac) is implicated in chromatin landscape regulation, impacting genomic stability, yet the effect of H3K56Ac on DNA base excision repair (BER) remains unclear. We determined whether H3K56Ac plays a role in regulating AP site incision by AP endonuclease 1 (APE1), an early step in BER.

A cassette of basic amino acids in histone H2B regulates nucleosome dynamics and access to DNA damage.

Nucleosome dynamics, such as spontaneous DNA unwrapping, are postulated to have a critical role in regulating the access of DNA repair machinery to DNA lesions within nucleosomes. However, the specific histone domains that regulate nucleosome dynamics and the impact of such changes in intrinsic nucleosome dynamics on DNA repair are not well understood. Previous studies identified a highly conserved region in the N-terminal tail of histone H2B known as the istone H2epression (or HBR) domain, which has a significant influence on gene expression, chromatin assembly, and DNA damage formation and repair.

Unencumbered Pol β lyase activity in nucleosome core particles.

Packaging of DNA into the nucleosome core particle (NCP) is considered to exert constraints to all DNA-templated processes, including base excision repair where Pol β catalyzes two key enzymatic steps: 5'-dRP lyase gap trimming and template-directed DNA synthesis. Despite its biological significance, knowledge of Pol β activities on NCPs is still limited. Here, we show that removal of the 5'-dRP block by Pol β is unaffected by NCP constraints at all sites tested and is even enhanced near the DNA ends.

DNA polymerase β uses its lyase domain in a processive search for DNA damage.

DNA polymerase (Pol) β maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol β must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol β are largely unknown, but are critical for understanding the fundamental features of the BER pathway.

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine.

Numerous ribonucleotides are incorporated into the genome during DNA replication. Oxidized ribonucleotides can also be erroneously incorporated into DNA. Embedded ribonucleotides destabilize the structure of DNA and retard DNA synthesis by DNA polymerases (pols), leading to genomic instability.

Site-specific Acetylation of Histone H3 Decreases Polymerase β Activity on Nucleosome Core Particles in Vitro.

Histone posttranslational modifications have been associated with changes in chromatin structure necessary for transcription, replication, and DNA repair. Acetylation is one of the most studied and best characterized histone posttranslational modifications, but it is not known if histone acetylation modulates base excision repair of DNA lesions in chromatin. To address this question, we generated nucleosome core particles (NCPs) containing site-specifically acetylated H3K14 or H3K56 and measured repair of uracil and single-nucleotide gaps.

Accessing DNA damage in chromatin: Preparing the chromatin landscape for base excision repair.

DNA damage in chromatin comes in many forms, including single base lesions that induce base excision repair (BER). We and others have shown that the structural location of DNA lesions within nucleosomes greatly influences their accessibility to repair enzymes. Indeed, a difference in the location of uracil as small as one-half turn of the DNA backbone on the histone surface can result in a 10-fold difference in the time course of its removal in vitro.

The structural location of DNA lesions in nucleosome core particles determines accessibility by base excision repair enzymes.

Background: Base excision repair is hindered by nucleosomes.

Results: Outwardly oriented uracils near the nucleosome center are efficiently cleaved; however, polymerase β is strongly inhibited at these sites.

Conclusion: The histone octamer presents different levels of constraints on BER, dependent on the structural requirements for enzyme activity.

Rotational dynamics of DNA on the nucleosome surface markedly impact accessibility to a DNA repair enzyme.

Histones play a crucial role in the organization of DNA in the nucleus, but their presence can prevent interactions with DNA binding proteins responsible for repair of DNA damage. Uracil is an abundant mutagenic lesion recognized by uracil DNA glycosylase (UDG) in the first step of base excision repair (BER). In nucleosome core particles (NCPs), we find substantial differences in UDG-directed cleavage at uracils rotationally positioned toward (U-In) or away from (U-Out) the histone core, or midway between these orientations (U-Mid).

Individual and population effects of eugregarine, Gregarina niphandrodes (Eugregarinida: Gregarinidae), on Tenebrio molitor (Coleoptera: Tenebrionidae).

Gregarines are single-celled parasites in the phylum Apicomplexa that infect invertebrates. They are highly abundant on three levels: among a large diversity of invertebrates, in the proportion of population of organisms they infect, and within individually infected organisms. Because of their remarkable prevalence, we hypothesize that they play an important role in support of their hosts.