Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and reconstitution experiments, we have discovered a major sub-pathway of conventional long-patch BER that involves formation of a 9-nucleotide gap 5' to the lesion. This new sub-pathway is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 poly(ADP-ribose) polymerase and RPA The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto-(ADP-ribosyl)ation and the choice between long-patch and single-nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long-patch BER and a new key regulation point for pathway choice in BER.

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Background: hMPG initiates repair of mutagenic and tumorigenic modified purine bases. Results: Two germ line hMPG variants showed reduced activity due to low affinity for DNA and facilitated lesion-induced mutations. Conclusion: Changes in amino acid sequence alter the function of hMPG, leading to genomic instability. Significance: Individuals possessing these hMPG variants may be at higher risk for genomic instability-related diseases.

Repair of oxidative stress- and inflammation-induced DNA lesions by the base excision repair (BER) pathway prevents mutation, a form of genomic instability which is often observed in cancer as ‘mutation hotspots’. This suggests that some sequences have inherent mutability, possibly due to sequence-related differences in repair. This study has explored intrinsic mutability as a consequence of sequence-specific repair of lipid peroxidation-induced DNA adduct, 1, N6-ethenoadenine (εA). For the first time, we observed significant delay in repair of ϵA at mutation hotspots in the tumor suppressor gene p53 compared to non-hotspots in live human hepatocytes and endothelial cells using an in-cell real time PCR-based method. In-cell and in vitro mechanism studies revealed that this delay in repair was due to inefficient turnover of N-methylpurine-DNA glycosylase (MPG), which initiates BER of εA. We determined that the product dissociation rate of MPG at the hotspot codons was ≈5–12-fold lower than the non-hotspots, suggesting a previously unknown mechanism for slower repair at mutation hotspots and implicating sequence-related variability of DNA repair efficiency to be responsible for mutation hotspot signatures.

Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50 = 2.6 µM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavanoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease.

DNA-protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA-protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

Reliable analytical modeling of the non-linear power spectrum (PS) of matter perturbations is among the chief pre-requisites for cosmological analyses from the largest sky surveys. This is especially true for the models that extend the standard general-relativity paradigm by adding the fifth force, where numerical simulations can be prohibitively expensive. Here we present a method for building accurate PS models for two modified gravity (MG) variants: namely the Hu-Sawicki f (R), and the normal branch of the Dvali-Gabadadze-Porrati (nDGP) braneworld. We start by modifying the standard halo model (HM) with respect to the baseline Lambda-Cold-Dark-Matter (ΛCDM) scenario, by using the HM components with specific MG extensions. We find that our P (k)HM retains 5% accuracy only up to mildly non-linear scales (k 0.3 h/ Mpc) when compared to PS from numerical simulations. At the same time, our HM prescription much more accurately captures the ratio Υ(k) = P (k)MG/P (k)ΛCDM up to non-linear scales. We show that using HM-derived Υ(k) together with a viable non-linear ΛCDM P (k) prescription (such as halofit), we render a much better and more accurate PS predictions in MG. The new approach yields considerably improved performance, with modeled P (k)MG being now accurate to within 5% all the way to non-linear scales of k 2.5 − 3 h/ Mpc. The magnitude of deviations from GR as fostered by these MG models is typically O(10%) in these regimes. Therefore reaching 5% PS modeling is enough for forecasting constraints on modern-era cosmological observables.

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