Description: Publication date: Available online 30 March 2018 Source: DNA Repair Author(s): Deniz Ceylan, Gamze Tuna, Güldal Kirkali, Zeliha Tunca, Güneş Can, Hidayet Ece Arat, Melis Kant, Miral Dizdaroglu, Ayşegül Özerdem Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder.

Description: Publication date: Available online 26 March 2018 Source: DNA Repair Author(s): Yong-Jie Deng, Lei Feng, Huan Zhou, Xiang Xiao, Feng-Ping Wang, Xi-Peng Liu In cells, degrading DNA and RNA by various nucleases is very important. These processes are strictly controlled and regulated to maintain DNA integrity and to mature or recycle various RNAs. NanoRNase (Nrn) is a 3′-exonuclease that specifically degrades nanoRNAs shorter than 5 nucleotides. Several Nrns have been identified and characterized in bacteria, mainly in Firmicutes. Archaea often grow in extreme environments and might be subjected to more damage to DNA/RNA, so DNA repair and recycling of damaged RNA are very important in archaea. There is no report on the identification and characterization of Nrn in archaea. Aeropyrum pernix encodes three potential Nrns: NrnA (Ape1437), NrnB (Ape0124), and an Nrn-like protein Ape2190. Biochemical characterization showed that only Ape0124 could degrade ssDNA and ssRNA from the 3′-end in the presence of Mn 2+ . Interestingly, unlike bacterial Nrns, Ape0124 preferred to ssDNA, including short nanoDNA, while degraded nanoRNA in lower efficiency. The 3′-DNA backbone was required for efficiently hydrolyzing the phosphodiester bonds. In addition, Ape0124 also degraded the 3′-overhang of double-stranded DNA. Interestingly, Ape0124 could hydrolyze pAp into AMP, which is a feature of bacterial NrnA, not NrnB. Our results indicate that Ape0124 is a novel Nrn with a combined substrate profile of bacterial NrnA and NrnB.

Description: Publication date: Available online 21 March 2018 Source: DNA Repair Author(s): Eloy Andres Perez-Yepez, Hector Ivan Saldivar-Ceron, Olga Villamar-Cruz, Carlos Perez-Plasencia, Luis Enrique Arias-Romero p21-activated kinase 1 (PAK1) is a serine/threonine kinase activated by the small GTPases Rac1 and Cdc42. It is located in the chromosome 11q13 and is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme plays a pivotal role in the control of a number of fundamental cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, it is well documented that PAK1 also plays crucial roles in the nucleus participating in mitotic events and gene expression through its association and/or phosphorylation of several transcription factors, transcriptional co-regulators and cell cycle-related proteins, including Aurora kinase A (AURKA), polo-like kinase 1 (PLK1), the forkhead transcription factor (FKHR), estrogen receptor α (ERα), and Snail. More recently, PAK signaling has emerged as a component of the DNA damage response (DDR) as PAK1 activity influences the cellular sensitivity to ionizing radiation and promotes the expression of several genes involved in the Fanconi Anemia/BRCA pathway. This review will focus on the nuclear functions of PAK1 and its role in the regulation of DNA damage repair.

Description: Publication date: Available online 21 March 2018 Source: DNA Repair Author(s): Jeffrey R. Whiteaker, Lei Zhao, Richard G. Ivey, Marilyn Sanchez-Bonilla, Heather D. Moore, Regine M. Schoenherr, Ping Yan, Chenwei Lin, Akiko Shimamura, Amanda G. Paulovich The Fanconi anemia pathway is an important coordinator of DNA repair pathways and is particularly relevant to repair of DNA inter-strand crosslinks. Central to the pathway is monoubiquitination of FANCD2, requiring the function of multiple proteins in an upstream Fanconi core complex. We present development and analytical characterization of a novel assay for quantification of unmodified and monoubiquitinated FANCD2 proteoforms, based on peptide immunoaffinity enrichment and targeted multiple reaction monitoring mass spectrometry (immuno-MRM). The immuno-MRM assay is analytically characterized using fit-for-purpose method validation. The assay linear range is >3 orders of magnitude with total repeatability 〈16% CV. In proof-of-principle experiments, we demonstrate application of the multiplex assay by quantifying the FANCD2 proteoforms following mitomycin-c treatment in an isogenic pair of FancA -corrected and uncorrected cell lines, as well as primary peripheral blood mononuclear cells from Fanconi Anemia patients. Additionally, we demonstrate detection of endogenous FANCD2 monoubiquitination in human breast cancer tissue. The immuno-MRM assay provides a potential functional diagnostic for patients with Fanconi Anemia with defects in the upstream FA complex or FANCD2, and a potential test for predicting sensitivity to DNA cross-linking agents in human cancers. Graphical abstract

Description: Publication date: April 2018 Source: DNA Repair, Volume 64

Description: Publication date: Available online 8 March 2018 Source: DNA Repair Author(s): Yuqin Cai, Iwen Fu, Nicholas E. Geacintov, Yingkai Zhang, Suse Broyde How DNA lesions in nucleosomes are recognized for global genome nucleotide excision repair (GG-NER) remains poorly understood, and the roles that histone tails may play remains to be established. Histone H3 and H4 N-terminal tails are of particular interest as their acetylation states are important in regulating nucleosomal functions in transcription, replication and repair. In particular the H3 tail has been the focus of recent attention as a site for the interaction with XPC, the GG-NER lesion recognition factor. Here we have investigated how the structure and dynamics of the DNA lesion cis -B[ a ]P-dG, derived from the environmental carcinogen benzo[ a ]pyrene (B[ a ]P), is impacted by the presence of flanking H3 and H4 tails. This lesion is well-repaired by GG-NER, and adopts a base-displaced/intercalated conformation in which the lesion partner C is displaced into the major groove. We used molecular dynamics simulations to obtain structural and dynamic characterizations for this lesion positioned in nucleosomal DNA so that it is bracketed by the H3 and H4 tails. The H4 tail was studied in unacetylated and acetylated states, while the H3 tail was unacetylated, its state when it binds XPC (Kakumu, Nakanishi et al., 2017). Our results reveal that upon acetylation, the H4 tail is released from the DNA surface; the H3 tail then forms a pocket that induces flipping and capture of the displaced lesion partner base C. This reveals synergistic effects of the behavior of the two tails. We hypothesize that the dual capability of the H3 tail to sense the displaced lesion partner base and to bind XPC could foster recognition of this lesion by XPC for initiation of GG-NER in nucleosomes. Graphical abstract

Description: Publication date: Available online 7 March 2018 Source: DNA Repair Author(s): Jinyu Wang, Haitao Zhang, Mohammed Al-Shibar, Belinda Willard, Alo Ray, Kurt W. Runge Telomeres, the ends of eukaryotic chromosomes, consist of repetitive DNA sequences and their bound proteins that protect the end from the DNA damage response. Short telomeres with fewer repeats are preferentially elongated by telomerase. Tel1, the yeast homolog of human ATM kinase, is preferentially recruited to short telomeres and Tel1 kinase activity is required for telomere elongation. Rif1, a telomere-binding protein, negatively regulates telomere length by forming a complex with two other telomere binding proteins, Rap1 and Rif2, to block telomerase recruitment. Rif1 has 14 SQ/TQ consensus phosphorylation sites for ATM kinases, including 6 in a SQ/TQ Cluster Domain (SCD) similar to other DNA damage response proteins. These 14 sites were analyzed as N-terminal, SCD and C-terminal domains. Mutating some sites to non-phosphorylatable residues increased telomere length in cells lacking Tel1 while a different set of phosphomimetic mutants increased telomere length in cells lacking Rif2, suggesting that Rif1 phosphorylation has both positive and negative effects on length regulation. While these mutations did not alter the sensitivity to DNA damaging agents, inducing telomere-specific damage by growing cells lacking YKU70 at high temperature revealed a role for the SCD. Mass spectrometry of Rif1 from wild type cells or those induced for telomere-specific DNA damage revealed increased phosphorylation in cells with telomere damage at an ATM consensus site in the SCD, S1351, and non-ATM sites S181 and S1637. A phosphomimetic rif1-S1351E mutation caused an increase in telomere length at synthetic telomeres but not natural telomeres. These results indicate that the Rif1 SCD can modulate Rif1 function. As all Rif1 orthologs have one or more SCD domains, these results for yeast Rif1 have implications for the regulation of Rif1 function in humans and other organisms.

Description: Publication date: Available online 7 March 2018 Source: DNA Repair Author(s): Pingping Jia, Weihang Chai Genome instability gives rise to cancer. MLH1, commonly known for its important role in mismatch repair (MMR), DNA damage signaling and double-strand break (DSB) repair, safeguards genome stability. Recently we have reported a novel role of MLH1 in preventing aberrant formation of interstitial telomeric sequences (ITSs) at intra-chromosomal regions. Deficiency in MLH1, in particular its N-terminus, leads to an increase of ITSs. Here, we identify that the ATPase activity in the MLH1 N-terminal domain is important for suppressing the formation of ITSs. The ATPase activity is also needed for recruiting MLH1 to DSBs. Moreover, defective ATPase activity of MLH1 causes an increase in micronuclei formation. Our results highlight the crucial role of MLH1’s ATPase domain in preventing the aberrant formation of telomeric sequences at the intra-chromosomal regions and preserving genome stability.

Description: Publication date: Available online 6 March 2018 Source: DNA Repair Author(s): Nidhi Sharma, Srinivas Chakravarthy, Matthew J. Longley, William C. Copeland, Aishwarya Prakash The 16.5 kb mitochondrial genome is subjected to damage from reactive oxygen species (ROS) generated in the cell during normal cellular metabolism and external sources such as ionizing radiation and ultraviolet light. ROS cause harmful damage to DNA bases that could result in mutagenesis and various diseases, if not properly repaired. The base excision repair (BER) pathway is the primary pathway involved in maintaining the integrity of mtDNA. Several enzymes that partake in BER within the nucleus have also been identified in the mitochondria. The nei-like (NEIL) DNA glycosylases initiate BER by excising oxidized pyrimidine bases and others such as the ring-opened formamidopyrimidine and the hydantoin lesions. During BER, the NEIL enzymes interact with proteins that are involved with DNA replication and transcription. In the current manuscript, we detected NEIL1 in purified mitochondrial extracts from human cells and showed that NEIL1 interacts with the human mitochondrial single-stranded DNA binding protein (mtSSB) via its C-terminal tail using protein painting, far-western analysis, and gel-filtration chromatography. Finally, we scrutinized the NEIL1-mtSSB interaction in the presence and absence of a partial-duplex DNA substrate using a combination of multi-angle light scattering (MALS) and small-angle X-ray scattering (SAXS). The data indicate that NEIL1 and homotetrameric mtSSB form a larger ternary complex in presence of DNA, however, the tetrameric form of mtSSB gets disrupted by NEIL1 in the absence of DNA as revealed by the formation of a smaller NEIL1-mtSSB monomer complex.

Description: Publication date: Available online 5 March 2018 Source: DNA Repair Author(s): Teresa Morales-Ruiz, Álvaro C. Romero-Valenzuela, Vanessa M. Vázquez-Grande, Teresa Roldán-Arjona, Rafael R. Ariza, Dolores Córdoba-Cañero Base excision repair (BER) is a major defense pathway against spontaneous DNA damage. This multistep process is initiated by DNA glycosylases that recognise and excise the damaged base, and proceeds by the concerted action of additional proteins that perform incision of the abasic site, gap filling and ligation. BER has been extensively studied in bacteria, yeasts and animals. Although knowledge of this pathway in land plants is increasing, there are no reports detecting BER in algae. We describe here an experimental in vitro system allowing the specific analysis of BER in the model alga Chlamydomonas reinhardtii . We show that C. reinhardtii cell-free extracts contain the enzymatic machinery required to perform BER of ubiquitous DNA lesions, such as uracil and abasic sites. Our results also reveal that repair can occur by both single-nucleotide insertion and long-patch DNA synthesis. The experimental system described here should prove useful in the biochemical and genetic dissection of BER in algae, and may contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways in photosynthetic eukaryotes.