Advances in Understanding the Complex Mechanisms of DNA Interstrand Cross-Link Repair

DNA interstrand cross-links (ICLs) are lesions caused by a variety of endogenous metabolites, environmental exposures, and cancer chemotherapeutic agents that have two reactive groups. The common feature of these diverse lesions is that two nucleotides on opposite strands are covalently joined. ICLs prevent the separation of two DNA strands and therefore essential cellular processes including DNA replication and transcription. ICLs are mainly detected in S phase when a replication fork stalls at an ICL. Damage signaling and repair of ICLs are promoted by the Fanconi anemia pathway and numerous posttranslational modifications of DNA repair and chromatin structural proteins. ICLs are also detected and repaired in nonreplicating cells, although the mechanism is less clear. A unique feature of ICL repair is that both strands of DNA must be incised to completely remove the lesion. This is accomplished in sequential steps to prevent creating multiple double-strand breaks. Unhooking of an ICL from one strand is followed by translesion synthesis to fill the gap and create an intact duplex DNA, harboring a remnant of the ICL. Removal of the lesion from the second strand is likely accomplished by nucleotide excision repair. Inadequate repair of ICLs is particularly detrimental to rapidly dividing cells, explaining the bone marrow failure characteristic of Fanconi anemia and why cross-linking agents are efficacious in cancer therapy. Herein, recent advances in our understanding of ICLs and the biological responses they trigger are discussed.clos

PARP Inhibition in Prostate Cancer With Homologous Recombination Repair Alterations

Purpose: With the broad use of next-generation sequencing assays, it has become clear that mutations in DNA repair genes are more commonly found than previously reported. In advanced prostate cancer patients with BRCA1/2 or ATM mutations, poly (ADP-ribose) polymerase inhibition (PARPi) causes an increased overall survival advantage compared with patients without these mutations. This review explores the advantages and limitations of PARPi treatment and its use beyond BRCA1/2-altered tumors. Furthermore, it discusses the benefits of current biomarkers and what role functional biomarkers and organoids may play in addressing the involvement of homologous recombination repair mutations in tumor development and progression. Methods: A systematic review was conducted in MEDLINE, National Library of Medicine, and ClinicalTrials.gov to identify studies published between January 1, 2016, and August 31, 2021. The search strategy incorporated terms for PARPi, BRCA, DNA damage, homologous recombination, organoids, patient-derived organoids, biomarker AND prostate cancer, breast cancer, ovarian cancer. Results: A total of 261 records remained after duplicate removal, 69 of which were included in the qualitative synthesis. Conclusion: To improve the outcome of targeted therapy and increase sensitivity of tumor detection, patients should be repeatedly screened for DNA repair gene alterations and biomarkers. Future clinical studies should explore the use of PARPi beyond BRCA1/2 mutations and focus on finding new synthetically lethal interactions

Iron-coated polymer films with high antibacterial activity under indoor and outdoor light, prepared by different facile pre-treatment and deposition methods

In the present work, we study the fabrication of self-cleaning antibacterial surfaces, active under indoor or outdoor light. In order to obtain the highest activity with the lowest complexity we assess mild conditions of preparation (temperatures), pre-treatment (physical, chemical) and deposition method (dip coating, spraying). We report that the combination of deposition time and temperature during this phase on PET can lead to effective germicidal films (< 40 min under solar light) and pre-treatment with simple sandpaper scratching or acetone dissolution increase the inactivation kinetics more than 35%. Spray coating always led to higher germicidal efficiencies, due to the differentiated layering during deposition, reaching total inactivation under either indoor or solar light. Furthermore, the non-pretreated films were very robust over 10 re-uses and the pre-treated ones led to virtually no loss of antibacterial activity. Other materials such as polyurethane (PU) and LDPE were effectively used, with pre-treated PU reaching the fastest inactivation (< 30 min). Finally, the costly Fe reagent was effectively replaced with natural Fe oxides, which were equally efficient in pre-treated surfaces. In overall, compared to real world conditions, a very high microbial load was eliminated, in either indoor or outdoor environments, meeting the demands where the infection problems are high and the means are scarce

Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV-induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPFR153P) were compared to an XP-causing mutation (XPFR799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPFR153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPFR153P-ERCC1 into the nucleus of XPF-deficient human cells restored nucleotide excision repair of UV induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex*

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (pol β), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol β is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol β or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol β with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a “hit and run” mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process