Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.

Unique structural features of mammalian NEIL2 DNA glycosylase prime its activity for diverse DNA substrates and environments

SUMMARYOxidative damage on DNA arising from both endogenous and exogenous sources can result in base modifications that promote errors in replication as well as generate sites of base loss (abasic sites) that present unique challenges to maintaining genomic integrity. These lesions are excised by DNA glycosylases in the first step of the base excision repair pathway. Here we present the first crystal structure of a NEIL2 glycosylase, an enzyme active on cytosine oxidation products and abasic sites. The structure reveals an unusual “open” conformation not seen in NEIL1 or NEIL3 orthologs. NEIL2 is predicted to adopt a “closed” conformation when bound to its substrate. Combined crystallographic and solution scattering studies show the enzyme to be conformationally dynamic in a manner distinct among the NEIL glycosylases and provide insight into the unique substrate preference of the enzyme. Additionally, we characterized three cancer variants of human NEIL2, namely S140N, G230W, and G303R.

Macrophages Promote DNA Repair of Double Strand Break in Multiple Myeloma Cells By Non-Homologous End Joining(NHEJ), Nevertheless Decrease Its Accuracy

Multiple myeloma (MM) is a hematological malignancy of B cells, characterized by clonal proliferation of malignant plasma cells. DNA damage and genomic instability play an important role in the pathogenesis of MM. Based on the characteristics of high heterogeneity and genomic instability of MM, and the protective effect of MΦs on MM cells (MMCs), our study intended to further clarify whether MΦs affect MMCs DNA damage response (DDR) and DNA repair, and the relationship between MΦs and genomic instability of MMCs. We found that the content of MΦs in bone marrow biopsy of MM patients was related to the results of cytogenetics (tested by FISH). The higher the content of MΦs, the more complicated the cytogenetic abnormalities of patients, especially in the IgH translocation, D13S319 locus deletion and RB1 deletion. In our study, MΦs were harvested from peripheral blood monocytes (PBMCs) , which were incubated for 7 days with M-CSF. Flow cytometry was used to detect M-CSF induced macrophages (MΦs) in vitro, and CD163 and CD206 were highly expressed in MΦs which implied that MΦs tended to be M2 type. The incubated MΦs were used in the following experiments. Our study showed that MΦs reduced the baseline γH2AX of MMCs, and contributed to MMCs surviving in the case of genomic instability detected by Western blot and immunofluorescence. We also confirmed that MΦs contribute to repairing the DNA damage in myeloma cells with the methods of comet assay. In the case of severe injury of MMCs' DNA, MΦs promoted the DDR and DNA damage repair. We examined the effects of macrophages on HR and NHEJ using U2OS cells. HR repair was measured in U2OS-HR cells loaded with SCR reporter (HR reporter) while NHEJ repair was measured in U2OS-NHEJ cells loaded with vGEJ reporter (NHEJ Reporter). We found that macrophages increased NHEJ but had no sense on total HR. In order to detect NHEJ level in endogenous genes, we adopted paired gRNA-CRISPR/Cas9 system. AAVS1 and HBB were used as detection genes, and the sequence of about 250 bp near the NHEJ interface was sequenced by NGS. The results proclaimed that the MΦs co-culture group significantly increased the efficiency of NHEJ, and decreased proportion of accurate NHEJ repair. In addition, analysis of the length of base sequence loss showed that the probability of base loss >3 bp in the MΦs co-culture group was higher than that MMCs in the group cultured alone. In the HBB site, MΦs also prolonged the average length of base loss in NHEJ. Furthermore, we used gRNA-CRISPR/Cas9 technology to cause fixed-point cleavage in AAVS1 and HBB respectively, and detected translocation by PCR and NGS with comparing the translocation reads between different groups. The results showed that MΦs promoted the probability of chromosomal translocation, which was of great importance in MM's occurrence and progression. Disclosures No relevant conflicts of interest to declare.

Pure-Tone–Spondee Threshold Relationships in Functional Hearing Loss: A Test of Loudness Contribution

Purpose The purpose of this article is to examine explanations for pure-tone average–spondee threshold differences in functional hearing loss. Method Loudness magnitude estimation functions were obtained from 24 participants for pure tones (0.5 and 1.0 kHz), vowels, spondees, and speech-shaped noise as a function of level (20–90 dB SPL). Participants listened monaurally through earphones. Loudness predictions were obtained for the same stimuli by using a computational, dynamic loudness model. Results When evaluated at the same SPL, speech-shaped noise was judged louder than vowels/spondees, which were judged louder than tones. Equal-loudness levels were inferred from fitted loudness functions for the group. For the clinical application, the 2.1-dB difference between spondees and tones at equal loudness became a 12.1-dB difference when the stimuli were converted from SPL to HL. Conclusions Nearly all of the pure-tone average–spondee threshold differences in functional hearing loss are attributable to references for calibration for 0 dB HL for tones and speech, which are based on detection and recognition, respectively. The recognition threshold for spondees is roughly 9 dB higher than the speech detection threshold; persons feigning a loss, who base loss magnitude on loudness, do not consider this difference. Furthermore, the dynamic loudness model was more accurate than the static model.