Active turnover of genomic methylcytosine in pluripotent cells.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.

TENT4A Non-Canonical Poly(A) Polymerase Regulates DNA-Damage Tolerance via Multiple Pathways That Are Mutated in Endometrial Cancer.

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

Molecular mechanisms of xeroderma pigmentosum (XP) proteins.

Nucleotide excision repair (NER) is a highly versatile and efficient DNA repair process, which is responsible for the removal of a large number of structurally diverse DNA lesions. Its extreme broad substrate specificity ranges from DNA damages formed upon exposure to ultraviolet radiation to numerous bulky DNA adducts induced by mutagenic environmental chemicals and cytotoxic drugs used in chemotherapy. Defective NER leads to serious diseases, such as xeroderma pigmentosum (XP). Eight XP complementation groups are known of which seven (XPA-XPG) are caused by mutations in genes involved in the NER process. The eighth gene, XPV, codes for the DNA polymerase ɳ, which replicates through DNA lesions in a process called translesion synthesis (TLS). Over the past decade, detailed structural information of these DNA repair proteins involved in eukaryotic NER and TLS have emerged. These structures allow us now to understand the molecular mechanism of the NER and TLS processes in quite some detail and we have begun to understand the broad substrate specificity of NER. In this review, we aim to highlight recent advances in the process of damage recognition and repair as well as damage tolerance by the XP proteins.

Structural Insights into the Recognition of N2 -Aryl- and C8-Aryl DNA Lesions by the Repair Protein XPA/Rad14.

Aromatic amines are strongly carcinogenic. They are activated in the liver to give reactive nitrenium ions that react with nucleobases within the DNA duplex. The reaction occurs predominantly at the C8 position of the dG base, thereby giving C8-acetyl-aryl- or C8-aryl-dG adducts in an electrophilic aromatic substitution reaction. Alternatively, reaction with the exocyclic 2-NH2 group is observed. Although the C8 adducts retain base-pairing properties, base pairing is strongly compromised in the case of the N2 adducts. Here we show crystal structures of two DNA lesions, N2 -acetylnaphthyl-dG and C8-fluorenyl-dG, within a DNA duplex recognized by the repair protein Rad14. The structures confirm that two molecules of the repair protein recognize the lesion and induce a 72 or 78° kink at the site of the damage. Importantly, the same overall kinked structure is induced by binding of the repair proteins, although the structurally different lesions result in distinct stacking interactions of the lesions within the duplex. The results suggest that the repair protein XPA/Rad14 is a sensor that recognizes flexibility. The protein converts the information that structurally different lesions are present in the duplex into a unifying sharply kinked recognition motif.

Quantitative LC-MS Provides No Evidence for m6 dA or m4 dC in the Genome of Mouse Embryonic Stem Cells and Tissues.

Until recently, it was believed that the genomes of higher organisms contain, in addition to the four canonical DNA bases, only 5-methyl-dC (m5 dC) as a modified base to control epigenetic processes. In recent years, this view has changed dramatically with the discovery of 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNA from stem cells and brain tissue. N6 -methyldeoxyadenosine (m6 dA) is the most recent base reported to be present in the genome of various eukaryotic organisms. This base, together with N4 -methyldeoxycytidine (m4 dC), was first reported to be a component of bacterial genomes. In this work, we investigated the levels and distribution of these potentially epigenetically relevant DNA bases by using a novel ultrasensitive UHPLC-MS method. We further report quantitative data for m5 dC, hmdC, fdC, and cadC, but we were unable to detect either m4 dC or m6 dA in DNA isolated from mouse embryonic stem cells or brain and liver tissue, which calls into question their epigenetic relevance.

Structural Basis for Bulky-Adduct DNA-Lesion Recognition by the Nucleotide Excision Repair Protein Rad14.

Heterocyclic aromatic amines react with purine bases and result in bulky DNA adducts that cause mutations. Such structurally diverse lesions are substrates for the nucleotide excision repair (NER). It is thought that the NER machinery recognises and verifies distorted DNA conformations, also involving the xeroderma pigmentosum group A and C proteins (XPA, XPC) that act as a scaffold between the DNA substrate and several other NER proteins. Here we present the synthesis of DNA molecules containing the polycyclic, aromatic amine C8-guanine lesions acetylaminophenyl, acetylaminonaphthyl, acetylaminoanthryl, and acetylaminopyrenyl, as well as their crystal structures in complex with the yeast XPA homologue Rad14. This work further substantiates the indirect lesion-detection mechanism employed by the NER system that recognises destabilised and deformable DNA structures.