LigD: A Structural Guide to the Multi-Tool of Bacterial Non-Homologous End Joining

DNA double-strand breaks are the most lethal form of damage for living organisms. The non-homologous end joining (NHEJ) pathway can repair these breaks without the use of a DNA template, making it a critical repair mechanism when DNA is not replicating, but also a threat to genome integrity. NHEJ requires proteins to anchor the DNA double-strand break, recruit additional repair proteins, and then depending on the damage at the DNA ends, fill in nucleotide gaps or add or remove phosphate groups before final ligation. In eukaryotes, NHEJ uses a multitude of proteins to carry out processing and ligation of the DNA double-strand break. Bacterial NHEJ, though, accomplishes repair primarily with only two proteins–Ku and LigD. While Ku binds the initial break and recruits LigD, it is LigD that is the primary DNA end processing machinery. Up to three enzymatic domains reside within LigD, dependent on the bacterial species. These domains are a polymerase domain, to fill in nucleotide gaps with a preference for ribonucleotide addition; a phosphoesterase domain, to generate a 3′-hydroxyl DNA end; and the ligase domain, to seal the phosphodiester backbone. To date, there are no experimental structures of wild-type LigD, but there are x-ray and nuclear magnetic resonance structures of the individual enzymatic domains from different bacteria and archaea, along with structural predictions of wild-type LigD via AlphaFold. In this review, we will examine the structures of the independent domains of LigD from different bacterial species and the contributions these structures have made to understanding the NHEJ repair mechanism. We will then examine how the experimental structures of the individual LigD enzymatic domains combine with structural predictions of LigD from different bacterial species and postulate how LigD coordinates multiple enzymatic activities to carry out DNA double-strand break repair in bacteria.

Comparing Effectiveness of Different Microhomology Lengths in Microhomology Mediated End Joining (MMEJ) Repair of DNA Double Stranded Breaks (DSBs)

DNA Double-Stranded Breaks (DSBs) are caused by genotoxic agents, such as ionizing radiation and chemical agents, and can cause an affected cell to undergo apoptosis or cell death. The process of microhomology-mediated end joining (MMEJ) shows promising results in the repair of DSBs in DNA. MMEJ is a mutagenic DSB repair mechanism that uses a certain length of homologous nucleotides adjacent to the DSB to align the broken DNA strands for repair. This can result in insertions, deletions, and even translocations of genes at the DSB site. This has led to discussions of debate on whether MMEJ is efficient in repairing DSBs in DNA. Based on the length of microhomology, the effectiveness of the DSB repair can vary. The purpose of this research is to examine MMEJ repair using micro-homologies of different lengths in Saccharomyces cerevisiae cells to test the effectiveness of MMEJ repair. The HIS3 gene located in chromosome 15 in the yeast cell is used to test for MMEJ repair, and the full microhomology length represents 311 base pairs (bp). Various crosses are performed on cells to attain desired genotypes that have the homologous chromosomes in alignment for MMEJ repair. After inducing DSBs, media-based testing is used for testing the efficiency of MMEj repair by checking for the presence of certain genes that may have formed or been deleted during the repair process.

Poly(ADP-Ribose) Polymerase-1 Inhibitors Drug Discovery, Design, and Development as Anticancer Agents from Past to Present: A Mini-Review

Cancer treatments are known for their life-threatening toxicities attributed to their low selectivity; hence, new therapeutic approaches are being developed as alternatives. Among those approaches is the DNA repair mechanism, where its inhibition results selectively in the death of cancerous cells. Poly(ADP-Ribose) Polymerase (PARP) is one of the enzymes involved in the repair of damaged DNA. The inhibition of PARP shows to be a promising approach for effective targeted treatment of cancer, especially in tumours with pre-existing Homologous-Repair (HR) defects (i.e., BRCA). Nicotinamide, which is one of the PARP catalytic products, was the first identified PARP inhibitor (PARPi). The first FDA-approved PARPi was Olaparib in 2014 for the treatment of BRCA mutated advanced ovarian cancer. Several clinical trials have been conducted to further improve PARPi. However, there are some concerns related to drug resistance, PARPi sensitive-tumour identification, and toxic accumulation of PARPi. This report will review the uses of PARPi, drug design and development of PARPi from past to present, current issues, and prospective plans.

A Review on Potential Antimutagenic Plants of Saudi Arabia

Mutagenic complications can cause disease in both present as well as future generations. The disorders are caused by exogenous and endogenous agents that damage DNA beyond the normal repair mechanism. Rapid industrialization and the population explosion have contributed immensely to changes in the environment, leading to unavoidable exposure to mutagens in our daily life. As it is impossible to prevent exposure, one of the better approaches is to increase the intake of anti-mutagenic substances derived from natural resources. This review summarizes some of the important plants in Saudi Arabia that might have the potential to exhibit anti-mutagenic activity. The data for the review were retrieved from Google scholar, NCBI, PUBMED, EMBASE and the Web of Science. The information in the study has importance since one of the major reasons for mutation is viral infection. Considering the pandemic situation due to novel coronavirus and its aftermath, the native plants of Saudi Arabia could become an important source for reducing mutagenic complications associated with exogenous agents, including viruses.

PARP dependent recruitment of RNA methylated at 8-adenosine is linked to base excision repair mechanism

Methylation of RNAs, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we observed that 8-methyladenosine (m8A)-modified RNA is recruited to UVA-microirradiated chromatin, which was reduced by inhibiting both DNA methylation and histone acetylation, especially in later phases of DDR. Most importantly, clinically used PARP inhibitor (PARPi), olaparib, prevents both m8A and m6A RNA accumulation at microirradiated chromatin. Testing the effect of PARPi on the efficiency of BER, NHEJ, and HR repair pathways, we observed that NHEJ repair proteins are down-regulated after PARP inhibition and recruitment of XRCC1, a factor of BER, to DNA lesions was abolished entirely. Conversely, the PARP inhibitor, olaparib, enhanced the genome-wide level of γH2AX that significantly interacted with m8A RNA, similar to DNA. Together, we showed that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent, similarly as XRCC1 playing a role in the BER mechanism. We found that γH2AX likely stabilizes m8A/m6A RNA-DNA hybrid loops that are formed during PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

Immunological considerations and vaccines against COVID-19

The outbreak COVID-19 is considered as a revolution in history of biological science. SARS-CoV-2 is a main cause of COVID-19 having resemblance with MERS-CoV and SARS-CoV. The response of host to the infection of SARS-CoV is multiform and strong. Initially, an effective host defense in the lung is affiliated with disease resolution and mild symptoms. The escaping of virus from immune response can lead to damage the alveoli, systematic inflammation, and ineffective lung repair mechanism with associated organ dysfunction. The immunological responses are necessary to fight with the virus and an effective and a safe vaccine is needed to overcome the pandemic. The development of vaccine is progressing fast, billions of dollars committed with more than 200 candidates before even knowing whether a vaccine candidate will succeed.

Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events

Background Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human. Results We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do. Conclusion Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

Structural insights into the repair mechanism of AGT for methyl-induced DNA damage

Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- and post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex.

A karboplatin-kemoterápia hatékonysága egy áttétes, kasztrációrezisztens, BRCA2-mutáció-pozitív prosztatarákos betegben

Összefoglaló. A sérült BRCA1/2 gént hordozó prosztatadaganatok klinikai szempontból elkülönülő, agresszív altípust képviselnek. Ugyanakkor a BRCA1/2 gén sérülése a DNS-támadáspontú kemoterápiákkal szemben érzékennyé teszi a daganatot, ami terápiás szempontból kihasználható. A platinaalapú kemoterápia hatékonysága prosztatarákban klinikai vizsgálatokkal nincs alátámasztva, ezért annak alkalmazására igen ritkán kerül sor. Közleményünkben egy előrehaladott stádiumú, agresszív prosztata adenocarcinomával diagnosztizált beteg esetét mutatjuk be, akinél a BRCA2-gén patogén mutációját találtuk, és akinél az előzőleg alkalmazott androgénmegvonásos, valamint docetaxelkezelések sikertelensége miatt karboplatinkezelést alkalmaztunk – ez a beteg állapotának, valamint radiológiai és biokémiai paramétereinek látványos javulásához vezetett. Ez az eset rámutat a DNS-hiba-javító mechanizmusban szerepet játszó gének terápiás szempontból történő felhasználásának potenciális előnyeire prosztatarákban. Orv Hetil. 2021; 162(25): 1004–1008. Summary. BRCA1/2 deficient prostate cancers represent a clinically distinct aggressive subtype. However, the presence of BRCA1/2 alterations enhance the sensitivity to platinum-based chemotherapies. The efficacy of platinum-based chemotherapies in prostate cancer has not been proven in prospective clinical studies and therefore these treatments are rarely used in prostate adenocarcinomas. Here we present a case of BRCA2 mutant prostate cancer, which was diagnosed at a metastatic stage and showed no or only little response to androgen deprivation and docetaxel therapies. Therefore, we started carboplatin chemotherapy which resulted in an exceptional response regarding biochemical, radiographic parameters accompanied by significant improvement of patients’ physical condition. This case underlines the potential therapeutic benefits of testing for genes involved in the DNA repair mechanism. Orv Hetil. 2021; 162(25): 1004–1008.