Ionic strength modulates excision of uracil by SMUG1 from nucleosome core particles.

Ionic strength affects many cellular processes including the packaging of genetic material in eukaryotes. For example, chromatin fibers are compacted in high ionic strength environments as are the minimal unit of packaging in chromatin, nucleosome core particles (NCPs). Furthermore, ionic strength is known to modulate several aspects of NCP dynamics including transient unwrapping of DNA from the histone protein core, nucleosome gaping, and intra- and internucleosomal interactions of the N-terminal histone tails. Changes in NCP structure may also impact interactions of transcriptional, repair, and other cellular machinery with nucleosomal DNA. One repair process, base excision repair (BER), is impacted by NCP structure and may be further influenced by changes in ionic strength. Here we examine the effects of ionic strength on the initiation of BER using biochemical assays. Using a population of NCPs containing uracil (U) at dozens of geometric locations, excision of U by single-strand selective monofunctional uracil DNA glycosylase (SMUG1) is assessed at higher and lower ionic strengths. SMUG1 has increased excision activity in the lower ionic strength conditions. On duplex DNA, however, SMUG1 activity is largely unaffected by ionic strength except at short incubation times, suggesting that changes in SMUG1 activity are likely due to alterations in NCP structure and dynamics. These results allow us to further understand the cellular role of SMUG1 in a changing ionic environment and broadly contribute to the understanding of BER on chromatin and genomic stability.

Histone variants H3.3 and H2A.Z/H3.3 facilitate excision of uracil from nucleosome core particles.

At the most fundamental level of chromatin organization, DNA is packaged as nucleosome core particles (NCPs) where DNA is wound around a core of histone proteins. This ubiquitous sequestration of DNA within NCPs presents a significant barrier to many biological processes, including DNA repair. We previously demonstrated that histone variants from the H2A family facilitate excision of uracil (U) lesions by DNA base excision repair (BER) glycosylases. Here, we consider how the histone variant H3.3 and double-variant H2A.Z/H3.3 modulate the BER enzymes uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1). Using an NCP model system with U:G base pairs at a wide variety of geometric positions we generate the global repair profile for both glycosylases. Enhanced excision of U by UDG and SMUG1 is observed with the H3.3 variant. We demonstrate that these H3.3-containing NCPs form two species: (1) octasomes, which contain the full complement of eight histone proteins and (2) hexasomes which are sub-nucleosomal particles that contain six histones. Both the octasome and hexasome species facilitate excision activity of UDG and SMUG1, with the largest impacts observed at sterically-occluded lesion sites and in terminal regions of DNA of the hexasome that do not closely interact with histones. For the double-variant H2A.Z/H3.3 NCPs, which exist as octasomes, the global repair profile reveals that UDG but not SMUG1 has increased U excision activity. The enhanced glycosylase activity reveals potential functions for these histone variants to facilitate BER in packaged DNA and contributes to our understanding of DNA repair in chromatin and its significance regarding mutagenesis and genomic integrity.

1,N6-Ethenoadenine: From Molecular to Biological Consequences†.

Genomic DNA is chemically reactive and therefore susceptible to damage by many exogenous and endogenous sources. Lesions produced from these damaging events can have various mutagenic and genotoxic consequences. This Perspective follows the journey of one particular lesion, 1,N6-ethenoadenine (εA), from its formation to replication and repair, and its role in cancerous tissues and inflammatory diseases. εA is generated by the reaction of adenine (A) with vinyl chloride or lipid peroxidation products. We present the miscoding properties of εA with an emphasis on how bacterial and mammalian cells can process lesions differently, leading to varied mutational spectra. But with information from these assays, we can better understand how the miscoding properties of εA lead to biological consequences and how genomic stability can be maintained via DNA repair mechanisms. We discuss how base excision repair (BER) and direct reversal repair (DRR) can minimize the biological consequences of εA lesions. Kinetic parameters of glycosylases and AlkB family enzymes are described, along with a discussion of the relative contributions of the BER and DRR pathways in the repair of εA. Because eukaryotic DNA is packaged in chromatin, we also discuss the impact of this packaging on BER and DRR, specifically in regards to repair of εA. Studying DNA lesions like εA in this context, from origin to biological implications, can provide crucial information to better understand prevention of mutagenesis and cancer.