Spontaneous deamination of cytosine and 5-methylcytosine residues in DNA and replacement of 5-methylcytosine residues with cytosine residues

Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25–200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r = 0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48 h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.

Exposure to genotoxic stress leads to the formation of various types of DNA damages that alter genome integrity. Importantly, DNA lesions occur at particular nucleotide sequences and their reactivity is also under the influence of genome compaction and epigenetic marks such as DNA methylation. The DNA repair pathways that are activated in response to DNA damages rely predominantly on de novo DNA synthesis, implying that the epigenomic landscape must also be accurately re-established. Therefore, complex interplays between base composition, DNA methylation level, and DNA repair pathways likely exist to efficiently maintain both genome and epigenome integrity at damaged and repaired genomic regions.

Base pair mismatches can erroneously be incorporated in the DNA. An adenine pairing with another adenine is one of the eight possible mismatches. The atomistic insights about the structure and dynamics of an A…A mismatch in a DNA (unbound form) is not yet accessible to any experimental technique. Earlier molecular dynamics (MD) simulations have shown that A…A mismatch in the midst of 5′CAG/3′GAC, 5′GAC/3′CAG and 5′CAA/3′GAT (underline represents the mismatch) are highly dynamic in nature. By employing MD simulation, the influence of an A…A mismatch in the midst of 5′GAA/3′CAT, 5′GAG/3′CAC, 5′AAC/3′TAG, 5′AAG/3′TAC, 5′TAA/3′AAT, 5′TAT/3′AAA and 5′AAT/3′TAA sequences have been investigated here. The results indicate that irrespective of the flanking sequences, the mismatch samples a variety of transient conformations, including a B-Z junction. Further, circular dichroism studies have been carried out to explore the ability of these sequences to bind with hZα_ADAR1 which specifically recognizes B-Z junction/Z-DNA. The results indicate that hZα_ADAR1 could not lead to a complete B to Z transition in the above sequences. Notably, a complete transition to Z-form has been reported earlier for 5′GAC/3′CAG upon titrating with hZα_ADAR1. Intriguingly, 5′AAC/3′TAG, 5′AAG/3′TAC and 5′GAA/3′CAT exhibit a B-Z junction formation rather than a complete transition to Z-form, similar to the situation of 5′CAA/3′GAT. These indicate that although A…A mismatch could induce a local B-Z junction transiently, hZα_ADAR1 requires the presence of a G…C/C…G base pair adjacent to the A…A mismatch for the binding. Additionally, the extent of B-Z junction has enhanced upon binding with hZα_ADAR1 in the presence of the A…A mismatch (specifically when CG, CA, AC, GA and AG steps occur), but not in the presence of the canonical base pairs. These confirm the inclination of A…A mismatch towards the B-Z junction.

Protein moonlighting is a phenomenon in which a single polypeptide chain can perform a number of different unrelated functions. Here we present our analysis of moonlighting in the case of selected DNA repair proteins which include G:T mismatch-specific thymine DNA glycosylase (TDG), methyl-CpG-binding domain protein 4 (MBD4), apurinic/apyrimidinic endonuclease 1 (APE1), AlkB homologs, poly (ADP-ribose) polymerase 1 (PARP-1) and single-strand selective monofunctional uracil DNA glycosylase 1 (SMUG1). Most of their additional functions are not accidental and clear patterns are emerging. Participation in RNA metabolism is not surprising as bases occurring in RNA are the same or very similar to those in DNA. Other common additional function involves regulation of transcription. This is not unexpected as these proteins bind to specific DNA regions for DNA repair, hence they can also be recruited to regulate transcription. Participation in demethylation and replication of DNA appears logical as well. Some of the multifunctional DNA repair proteins play major roles in many diseases, including cancer. However, their moonlighting might prove a major difficulty in the development of new therapies because it will not be trivial to target a single protein function without affecting its other functions that are not related to the disease.

Aging is driven by unavoidable entropic forces, physicochemical in nature, that damage the raw materials that constitute biological systems. Single cells experience and respond to stochastic physicochemical insults that occur either to the cells themselves or to their microenvironment, in a dynamic and reciprocal manner, leading to increased age-related cell-to-cell variation. We will discuss the biological mechanisms that integrate cell-to-cell variation across tissues resulting in stereotypical phenotypes of age.

In marine sediments, DNA occurs both inside and outside living organisms. DNA not enclosed in living cells may account for the largest fraction of total DNA, and include molecules locked within dead cells, organic and inorganic aggregates, adsorbed onto mineral matrices, and viral DNA. This DNA comprises genetic material released in situ from sediment microbial communities, as well as DNA of pelagic and terrestrial origin deposited to the seafloor. DNA not enclosed in living cells undermines the assumption of a direct link between the overall DNA pool and the local, currently living microbial assemblages, in terms of both microbial cell abundance and diversity. At the same time, the extracellular DNA may provide an integrated view of the biodiversity and ecological processes occurring on land, in marine water columns, and sediments themselves, thereby acting as an archive of genetic information which can be used to reconstruct past changes in source environments. In this review, we identify and discuss DNA pools in marine sediments, with special focus on DNA not enclosed in living cells, its origin, dynamics, and ecological and methodological implications. Achievements in deciphering the genetic information held within each DNA pool are presented along with still-standing challenges and major gaps in current knowledge.