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Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions.

Maintenance of genetic stability is of crucial importance for any form of life. Before cell division in each mammalian cell, the process of DNA replication must faithfully duplicate three billion bases with an absolute minimum of mistakes. This is complicated by the fact that DNA itself is highly reactive and is constantly attacked by endogenous and exogenous factors leading to 50,000-100,000 different damages in the DNA of human cells every day. In this mini-review we will focus on lesion bypass by DNA polymerase machines either in replication or repair, with particular focus on the repair of oxidative lesions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Here we investigated the ability of the human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg(2+) or Mn(2+). Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases beta and lambda was examined. The results show that (i) DNA polymerase lambda is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase lambda to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase lambda, (iv) DNA polymerase beta catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase beta. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucleotide at 2.0 Å resolution and reveals how Polλ performs replication across a DNA synapse joined by minimal base-pairing. Last, we find that Polλ is semisynthetic lethal with BRCA1 and BRCA2. Together, these studies indicate Polλ MMEJ as a distinct DSB repair mechanism., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols beta and lambda and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol lambda- dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol beta activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols lambda and beta.

We have very recently highlighted possible connections between DNA polymerases, the main enzymes in the DNA metabolism, and human diseases (Ramadan, K., Maga, G. and Hübscher, U.: DNA polymerases and diseases, In: Genome Integrity: Facets and Perspectives ed. Lankenau, D.-H. Springer Verlag, Heidelberg Germany, Vol 1, pp. 69-102, 2007). Beside a role in DNA replication of the genome DNA polymerases have fundamental functions in other aspect of DNA metabolism, such as DNA repair, DNA recombination, translesion DNA synthesis and cell cycle checkpoint. In the last decade many novel DNA polymerases have been identified, but their exact cellular functions still await clarification. We know that many DNA polymerases have redundant functions. It is a fact that specific inhibition of certain DNA polymerases is a promising approach to develop anticancer drugs. In this review we will concentrate on DNA repair proteins and translesion DNA polymerases as possible targets for anti cancer drugs.

It has been hypothesized that a replication associated repair pathway operates on base damage and single strand breaks (SSB) at replication forks. In this study, we present the isolation from the nuclei of human cycling cells of a multiprotein complex containing most of the essential components of base excision repair (BER)/SSBR, including APE1, UNG2, XRCC1 and POLbeta, DNA PK, replicative POLalpha, delta and epsilon, DNA ligase 1 and cell cycle regulatory protein cyclin A. Co-immunoprecipitation revealed that in this complex DNA repair proteins are physically associated to cyclin A and to DNA replication proteins including MCM7. This complex is endowed with DNA polymerase and protein kinase activity and is able to perform BER of uracil and AP sites. This finding suggests that a preassembled DNA repair machinery is constitutively active in cycling cells and is ready to be recruited at base damage and breaks occurring at replication forks.

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase alpha can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSalpha mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSalpha and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.

Human DNA polymerases (pols) beta and lambda could promote template slippage and generate -1 frameshifts on defined heteropolymeric DNA substrates containing a single abasic site. Kinetic data demonstrated that pol lambda was more efficient than pol beta in catalyzing translesion DNA synthesis past an abasic site, particularly in the presence of low nucleotide concentrations. Moreover, pol lambda was found to generate frameshifts in two ways: first, by using a nucleotide-stabilized primer misalignment mechanism, or second, by promoting primer reannealing using microhomology regions between the terminal primer sequence and the template strand. Our results suggest a molecular mechanism for the observed high in vivo rate of frameshifts generation by pol lambda and highlight the remarkable ability of pol lambda to promote microhomology pairing between two DNA strands, further supporting its proposed role in the nonhomologous end joining process.

Proliferating cell nuclear antigen (PCNA) was originally characterised as a DNA sliding clamp for replicative DNA polymerases and as an essential component of the eukaryotic chromosomal DNA replisome. Subsequent studies, however, have revealed its striking ability to interact with multiple partners, which are involved in several metabolic pathways, including Okazaki fragment processing, DNA repair, translesion DNA synthesis, DNA methylation, chromatin remodeling and cell cycle regulation. PCNA in mammalian cells thus appears to play a key role in controlling several reactions through the coordination and organisation of different partners. Two major questions have emerged: how do these proteins access PCNA in a coordinated manner, and how does PCNA temporally and spatially organise their functions? Structural and biochemical studies are starting to provide a first glimpse of how both tasks can be achieved.

Proliferating cell nuclear antigen (PCNA) has been shown to interact with a variety of DNA polymerases (pol) such as pol delta, pol epsilon, pol iota, pol kappa, pol eta, and pol beta. Here we show that PCNA directly interacts with the newly discovered pol lambda cloned from human cells. This interaction stabilizes the binding of pol lambda to the primer template, thus increasing its affinity for the hydroxyl primer and its processivity in DNA synthesis. However, no effect of PCNA was detected on the rate of nucleotide incorporation or discrimination efficiency by pol lambda. PCNA was found to stimulate efficient synthesis by pol lambda across an abasic (AP) site. When compared with pol delta, human pol lambda showed the ability to incorporate a nucleotide in front of the lesion. Addition of PCNA led to efficient elongation past the AP site by pol lambda but not by pol delta. However, when tested on a template containing a bulky DNA lesion, such as the major cisplatin Pt-d(GpG) adduct, PCNA could not allow translesion synthesis by pol lambda. Our results suggest that the complex between PCNA and pol lambda may play an important role in the bypass of abasic sites in human cells.

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.

Mfd couples transcription to nucleotide excision repair, and acts on RNA polymerases when elongation is impeded. Depending on impediment severity, this action results in either transcription termination or elongation rescue, which rely on ATP-dependent Mfd translocation on DNA. Due to its role in antibiotic resistance, Mfd is also emerging as a prime target for developing anti-evolution drugs. Here we report the structure of DNA-bound Mfd, which reveals large DNA-induced structural changes that are linked to the active site via ATPase motif VI. These changes relieve autoinhibitory contacts between the N- and C-termini and unmask UvrA recognition determinants. We also demonstrate that translocation relies on a threonine in motif Ic, widely conserved in translocases, and a family-specific histidine near motif IVa, reminiscent of the "arginine clamp" of RNA helicases. Thus, Mfd employs a mode of DNA recognition that at its core is common to ss/ds translocases that act on DNA or RNA.

Proliferating cell nuclear antigen is a marker of cell proliferation and genomic maintenance. The protein undergoes several post-translational modifications that expand its playground and amplify its functions. Ubiquitylation, sumoylation, acetylation, phosphorylation, and nitrosylation are some of the modifications that confer a plethora of protein interactions with the antigen. These interactions regulate abundant functions like DNA replication, DNA damage control, and cell cycle control. Due to the versatile functions of the antigen, it may serve as a marker or target for therapeutic intervention for various diseases. This protein can be used as a marker for studies done to validate growth in medical and dental fields. The growth and proliferation of cells is a necessary factor for cancer to progress, both at primary and metastatic sites. It was proposed that PCNA can aid in not only the identification but also as a therapeutic agent for the disease due to its role in cell proliferation and DNA damage control. This review expounds on the structure and functions of PCNA. It discusses how the interactions of the antigen with other proteins can be used to block or enhance the functions of the cell being commanded by the antigen. [ABSTRACT FROM AUTHOR]

The canonical role of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in repairing DNA double-strand breaks combined with its reported dysregulation in several malignancies has driven the development of DNA-PKcs inhibitors as therapeutics. However, until recently the relationship between DNA-PKcs and tumorigenesis has been primarily investigated with regard to its role in non-homologous end joining (NHEJ) repair. Emerging research has uncovered non-canonical DNA-PKcs functions involved with transcriptional regulation, telomere maintenance, metabolic regulation, and immune signaling all of which may also impinge on tumorigenesis. This review mainly discusses these non-canonical roles of DNA-PKcs in cellular biology and their potential contribution to tumorigenesis, as well as evaluating the implications of targeting DNA-PKcs for cancer therapy. [ABSTRACT FROM AUTHOR]

Replication protein A (RPA) is a heterotrimeric protein complex and the main single-stranded DNA (ssDNA)-binding protein in eukaryotes. RPA has key functions in most of the DNA-associated metabolic pathways and DNA damage signalling. Its high affinity for ssDNA helps to stabilise ssDNA structures and protect the DNA sequence from nuclease attacks. RPA consists of multiple DNA-binding domains which are oligonucleotide/oligosaccharide-binding (OB)-folds that are responsible for DNA binding and interactions with proteins. These RPA–ssDNA and RPA–protein interactions are crucial for DNA replication, DNA repair, DNA damage signalling, and the conservation of the genetic information of cells. Proteins such as ATR use RPA to locate to regions of DNA damage for DNA damage signalling. The recruitment of nucleases and DNA exchange factors to sites of double-strand breaks are also an important RPA function to ensure effective DNA recombination to correct these DNA lesions. Due to its high affinity to ssDNA, RPA's removal from ssDNA is of central importance to allow these metabolic pathways to proceed, and processes to exchange RPA against downstream factors are established in all eukaryotes. These faceted and multi-layered functions of RPA as well as its role in a variety of human diseases will be discussed. [ABSTRACT FROM AUTHOR]

Polyadenosine diphosphate-ribose polymerase (PARP) is a key modifying enzyme in cells, which participates in single-strand break repair and indirectly affects double-strand break repair. PARP inhibitors have shown great potential in oncotherapy by exploiting DNA damage repair pathways, and several small molecule PARP inhibitors have been approved by the U.S. Food and Drug Administration for treating various tumor types. PARP inhibitors not only have significant antitumor effects but also have some synergistic effects when combined with radiotherapy; therefore they have potential as radiation sensitizers. Here, we reviewed the advances and implications of PARP inhibitors in tumor radiotherapy sensitization. First, we summarized the multiple functions of PARP and the mechanisms by which its inhibitors exert antitumor effects. Next, we discuss the immunomodulatory effects of PARP and its inhibitors in tumors. Then, we described the theoretical basis of using PARP inhibitors in combination with radiotherapy and outlined their importance in oncological radiotherapy. Finally, we reviewed the current challenges in this field and elaborated on the future applications of PARP inhibitors as radiation sensitizers. A comprehensive understanding of the mechanism, optimal dosing, long-term safety, and identification of responsive biomarkers remain key challenges to integrating PARP inhibition into the radiotherapy management of cancer patients. Therefore, extensive research in these areas would facilitate the development of precision radiotherapy using PARP inhibitors to improve patient outcomes. [ABSTRACT FROM AUTHOR]

Base excision DNA repair (BER) is a key pathway safeguarding the genome of all living organisms from damage caused by both intrinsic and environmental factors. Most present knowledge about BER comes from studies of human cells, E. coli, and yeast. Plants may be under an even heavier DNA damage threat from abiotic stress, reactive oxygen species leaking from the photosynthetic system, and reactive secondary metabolites. In general, BER in plant species is similar to that in humans and model organisms, but several important details are specific to plants. Here, we review the current state of knowledge about BER in plants, with special attention paid to its unique features, such as the existence of active epigenetic demethylation based on the BER machinery, the unexplained diversity of alkylation damage repair enzymes, and the differences in the processing of abasic sites that appear either spontaneously or are generated as BER intermediates. Understanding the biochemistry of plant DNA repair, especially in species other than the Arabidopsis model, is important for future efforts to develop new crop varieties. [ABSTRACT FROM AUTHOR]

There is increasing evidence indicating the significant role of DDX5 (also called p68), acting as a master regulator and a potential biomarker and target, in tumorigenesis, proliferation, metastasis and treatment resistance for cancer therapy. However, DDX5 has also been reported to act as an oncosuppressor. These seemingly contradictory observations can be reconciled by DDX5's role in DNA repair. This is because cancer cell apoptosis and malignant transformation can represent the two possible outcomes of a single process regulated by DDX5, reflecting different intensity of DNA damage. Thus, targeting DDX5 could potentially shift cancer cells from a growth-arrested state (necessary for DNA repair) to apoptosis and cell killing. In addition to the increasingly recognized role of DDX5 in global genome stability surveillance and DNA damage repair, DDX5 has been implicated in multiple oncogenic signaling pathways. DDX5 appears to utilize distinct signaling cascades via interactions with unique proteins in different types of tissues/cells to elicit opposing roles (e.g., smooth muscle cells versus cancer cells). Such unique features make DDX5 an intriguing therapeutic target for the treatment of human cancers, with limited low toxicity to normal tissues. In this review, we discuss the multifaceted functions of DDX5 in DNA repair in cancer, immune suppression, oncogenic metabolic rewiring, virus infection promotion, and negative impact on the human microbiome (microbiota). We also provide new data showing that FL118, a molecular glue DDX5 degrader, selectively works against current treatment-resistant prostate cancer organoids/cells. Altogether, current studies demonstrate that DDX5 may represent a unique oncotarget for effectively conquering cancer with minimal toxicity to normal tissues. [ABSTRACT FROM AUTHOR]

Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases. [ABSTRACT FROM AUTHOR]

The comet assay in Drosophila has been used in the last few years to study DNA damage responses (DDR) in different repair-mutant strains and to compare them to analyze DNA repair. We have used this approach to study interactions between DNA repair pathways in vivo. Additionally, we have implemented an ex vivo comet assay, in which nucleoids from treated and untreated cells were incubated ex vivo with cell-free protein extracts from individuals with distinct repair capacities. Four strains were used: wild-type OregonK (OK), nucleotide excision repair mutant mus201, dmPolQ protein mutant mus308, and the double mutant mus201;mus308. Methyl methanesulfonate (MMS) was used as a genotoxic agent. Both approaches were performed with neuroblasts from third-instar larvae; they detected the effects of the NER and dmPolQ pathways on the DDR to MMS and that they act additively in this response. Additionally, the ex vivo approach quantified that mus201, mus308, and the double mutant mus201;mus308 strains presented, respectively, 21.5%, 52.9%, and 14.8% of OK strain activity over MMS-induced damage. Considering the homology between mammals and Drosophila in repair pathways, the detected additive effect might be extrapolated even to humans, demonstrating that Drosophila might be an excellent model to study interactions between repair pathways. [ABSTRACT FROM AUTHOR]

DNA–protein cross-links remain the least-studied type of DNA damage. Recently, their repair was shown to involve proteolysis; however, the fate of the peptide remnant attached to DNA is unclear. Particularly, peptide cross-links could interfere with DNA polymerases. Apurinuic/apyrimidinic (AP) sites, abundant and spontaneously arising DNA lesions, readily form cross-links with proteins. Their degradation products (AP site–peptide cross-links, APPXLs) are non-instructive and should be even more problematic for polymerases. Here, we address the ability of human DNA polymerases involved in DNA repair and translesion synthesis (POLβ, POLλ, POLη, POLκ and PrimPOL) to carry out synthesis on templates containing AP sites cross-linked to the N-terminus of a 10-mer peptide (APPXL-I) or to an internal lysine of a 23-mer peptide (APPXL-Y). Generally, APPXLs strongly blocked processive DNA synthesis. The blocking properties of APPXL-I were comparable with those of an AP site, while APPXL-Y constituted a much stronger obstruction. POLη and POLκ demonstrated the highest bypass ability. DNA polymerases mostly used dNTP-stabilized template misalignment to incorporate nucleotides when encountering an APPXL. We conclude that APPXLs are likely highly cytotoxic and mutagenic intermediates of AP site–protein cross-link repair and must be quickly eliminated before replication. [ABSTRACT FROM AUTHOR]

This article reviews the currently used therapeutic strategies to target DNA replication stress for cancer treatment in the clinic, highlighting their effectiveness and limitations due to toxicity and drug resistance. Cancer cells experience enhanced spontaneous DNA damage due to compromised DNA replication machinery, elevated levels of reactive oxygen species, loss of tumor suppressor genes, and/or constitutive activation of oncogenes. Consequently, these cells are addicted to DNA damage response signaling pathways and repair machinery to maintain genome stability and support survival and proliferation. Chemotherapeutic drugs exploit this genetic instability by inducing additional DNA damage to overwhelm the repair system in cancer cells. However, the clinical use of DNA-damaging agents is limited by their toxicity and drug resistance often arises. To address these issues, the article discusses a potential strategy to target the cancer-associated isoform of proliferating cell nuclear antigen (caPCNA), which plays a central role in the DNA replication and damage response network. Small molecule and peptide agents that specifically target caPCNA can selectively target cancer cells without significant toxicity to normal cells or experimental animals. [ABSTRACT FROM AUTHOR]

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3′ end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs—TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways. [ABSTRACT FROM AUTHOR]

DNA polymerase θ belongs to the A family of DNA polymerases and plays a key role in DNA repair and damage tolerance, including double-strand break repair and DNA translesion synthesis. Pol θ is often overexpressed in cancer cells and promotes their resistance to chemotherapeutic agents. In this review, we discuss unique biochemical properties and structural features of Pol θ, its multiple roles in protection of genome stability and the potential of Pol θ as a target for cancer treatment. [ABSTRACT FROM AUTHOR]

Sliding clamps play a pivotal role in the process of replication by increasing the processivity of the replicative polymerase. They also serve as an interacting platform for a plethora of other proteins, which have an important role in other DNA metabolic processes, including DNA repair. In other words, clamps have evolved, as has been correctly referred to, into a mobile "tool-belt" on the DNA, and provide a platform for several proteins that are involved in maintaining genome integrity. Because of the central role played by the sliding clamp in various processes, its study becomes essential and relevant in understanding these processes and exploring the protein as an important drug target. In this review, we provide an updated report on the functioning, interactions, and moonlighting roles of the sliding clamps in various organisms and its utilization as a drug target. [ABSTRACT FROM AUTHOR]

In response to DNA damage, cells have developed a sophisticated signaling pathway, consisting of DNA damage sensors, transducers, and effectors, to ensure efficient and proper repair of damaged DNA. During this process, posttranslational modifications (PTMs) are central events that modulate the recruitment, dissociation, and activation of DNA repair proteins at damage sites. Emerging evidence reveals that protein arginine methylation is one of the common PTMs and plays critical roles in DNA damage response. Protein arginine methyltransferases (PRMTs) either directly methylate DNA repair proteins or deposit methylation marks on histones to regulate their transcription, RNA splicing, protein stability, interaction with partners, enzymatic activities, and localization. In this review, we summarize the substrates and roles of each PRMTs in DNA damage response and discuss the synergistic anticancer effects of PRMTs and DNA damage pathway inhibitors, providing insight into the significance of arginine methylation in the maintenance of genome integrity and cancer therapies. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers worldwide. The occurrence of oncogenic KRAS mutations is considered a signature event in PDAC, leading to genomic instability. The aim of our study was to evaluate the impact of the oncogenic KRAS G12D mutation on the activity of the error-prone alt-EJ repair mechanism, and to investigate the potential role of Polθ in the development of pancreatic cancer. We found that oncogenic KRAS increases the expression of key alt-EJ proteins in a mouse and human PDAC model. Using TLR assay, we also found increased alt-EJ activity in mouse and human cell lines upon the expression of KRAS D12D. The inactivation/impairment of alt-EJ by polymerase theta (Polθ) depletion delays the development of pancreatic cancer and prolongs the survival of experimental mice, though it does not prevent the PDAC development, which leads to full-blown PDAC with disseminated metastasis. Our studies provide a high-value target as a novel therapeutic candidate for the treatment of pancreatic and other cancers. Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients. [ABSTRACT FROM AUTHOR]

The p21CDKN1A protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms. [ABSTRACT FROM AUTHOR]

Most of the neurodegenerative diseases and aging are associated with reactive oxygen species (ROS) or other intracellular damaging agents that challenge the genome integrity of the neurons. As most of the mature neurons stay in G0/G1 phase, replication-uncoupled DNA repair pathways including BER, NER, SSBR, and NHEJ, are pivotal, efficient, and economic mechanisms to maintain genomic stability without reactivating cell cycle. In these progresses, polymerases are prominent, not only because they are responsible for both sensing and repairing damages, but also for their more diversified roles depending on the cell cycle phase and damage types. In this review, we summarized recent knowledge on the structural and biochemical properties of distinct polymerases, including DNA and RNA polymerases, which are known to be expressed and active in nervous system; the biological relevance of these polymerases and their interactors with neuronal degeneration would be most graphically illustrated by the neurological abnormalities observed in patients with hereditary diseases associated with defects in DNA repair; furthermore, the vicious cycle of the trinucleotide repeat (TNR) and impaired DNA repair pathway is also discussed. Unraveling the mechanisms and contextual basis of the role of the polymerases in DNA damage response and repair will promote our understanding about how long-lived postmitotic cells cope with DNA lesions, and why disrupted DNA repair contributes to disease origin, despite the diversity of mutations in genes. This knowledge may lead to new insight into the development of targeted intervention for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The sliding clamp PCNA is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway. Focusing on Saccharomyces cerevisiae budding yeast, we summarized PCNA related-DDT and repair mechanisms that coordinately sustain genome stability and cell survival. In addition, we compared PCNA sequences from various fungal pathogens, considering recent advances in structural features. Importantly, the identification of PCNA epitopes may lead to potential fungal targets for antifungal drug development. [ABSTRACT FROM AUTHOR]

Background: Proliferating cell nuclear antigen (PCNA) is one of the key factors for the DNA replication process and DNA damage repair. Most proteins interacting with PCNA have a common binding motif: PCNA interacting protein box (PIP box). However, some proteins with non-canonical PIP-box have also been reported to be the key factors that interacted with PCNA. Results: Here we discovered the C terminal of a chromatin-remodeling factor CHR721 with non-canonical PIP-box was essential for interacting with OsPCNA in rice. Both OsPCNA and CHR721 were localized in the nuclei and function in response to DNA damages. Conclusions: Based on the results and previous work, we proposed a working model that CHR721 with non-canonical PIP-box interacted with OsPCNA and both of them probably participate in the DNA damage repair process. [ABSTRACT FROM AUTHOR]

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol d pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol d at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols δ, λ, β, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols β and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ-/- mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ. [ABSTRACT FROM AUTHOR]

Background: Emerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair. Methods and results: We used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of γΗ2ΑX foci, detected by immunofluorescence, and γH2AX levels detected by western blot analysis. After 24 h of cell recovery, γΗ2ΑX foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage. Conclusions: Taken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme. [ABSTRACT FROM AUTHOR]

DNA polymerases are important enzymes involved in DNA replication and repair. Based on sequence homology, DNA polymerases have been grouped into distinct families, which are A, B, X, and Y. The Pol X family consists of four members: Pol λ, μ, and β and terminal transferase or TdT. Members of the family X are involved in base excision repair, nonhomologous end joining (NHEJ), and V(D)J recombination. One of the most interesting pol X family members is DNA polymerase μ, discovered back in 2000. Subsequent studies established the importance of Pol μ as a repair polymerase in NHEJ and its interactions with the other proteins of the NHEJ machinery. Pol μ has a number of interesting properties, which sets it apart from the other known DNA polymerases, including its ability to synthesize DNA from an unpaired primer terminus as well in the complete absence of a template strand (terminal transferase activity). Another standout property of Pol μ is its reduced ability to discriminate between ribonucleotides and deoxyribonucleotides and its ability to utilize both ribonucleotides and deoxyribonucleotides as substrates during the gap‐filling stage of NHEJ. In this review, we provide a brief overview of Pol μ in double‐strand break repair and the current knowledge on its various functional aspects. [ABSTRACT FROM AUTHOR]

The GO system is part of the DNA base excision repair pathway and is required for the error-free repair of 8-oxoguanine (oxoG), one of the most common oxidative DNA lesions. Due to the ability of oxoG to form oxoG:A mispairs, this base is highly mutagenic. Its repair requires the action of two enzymes: 8-oxoguanine DNA glycosylase (Fpg or MutM in bacteria and OGG1 in eukaryotes), which removes oxoG from oxoG:C pairs, and adenine DNA glycosylase (MutY in bacteria and MUTYH in eukaryotes), which removes A from oxoG:A mispairs to prevent mutations. The third enzyme of the system (MutT in bacteria and MTH1 or NUDT1 in eukaryotes) hydrolyzes 8-oxo-2′-deoxyguanosine triphosphate, thus preventing its incorporation into DNA. Recent data point to the proteins of the GO system as promising targets for the therapy of cancer, autoimmune diseases, and bacterial infections. This review highlights the structure and specificity of the GO system in bacteria and eukaryotes and its unique role in mutation avoidance. [ABSTRACT FROM AUTHOR]

Summary: Gene editing and/or allele introgression with absolute precision and control appear to be the ultimate goals of genetic engineering. Precision genome editing in plants has been developed through various approaches, including oligonucleotide‐directed mutagenesis (ODM), base editing, prime editing and especially homologous recombination (HR)‐based gene targeting. With the advent of CRISPR/Cas for the targeted generation of DNA breaks (single‐stranded breaks (SSBs) or double‐stranded breaks (DSBs)), a substantial advancement in HR‐mediated precise editing frequencies has been achieved. Nonetheless, further research needs to be performed for commercially viable applications of precise genome editing; hence, an alternative innovative method for genome editing may be required. Within this scope, we summarize recent progress regarding precision genome editing mediated by microhomology‐mediated end joining (MMEJ) and discuss their potential applications in crop improvement. [ABSTRACT FROM AUTHOR]

About half the world's population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra‐erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localisation to the nuclear periphery, indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication, apoptosis and DNA repair pathways leading to cell death. Additionally, mislocalisation of the endogenously tagged protein confirmed its essential role in parasites' replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in P. falciparum. [ABSTRACT FROM AUTHOR]

Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome. [ABSTRACT FROM AUTHOR]

FAN1 encodes a DNA repair nuclease. Genetic deficiencies, copy number variants, and single nucleotide variants of FAN1 have been linked to karyomegalic interstitial nephritis, 15q13.3 microdeletion/microduplication syndrome (autism, schizophrenia, and epilepsy), cancer, and most recently repeat expansion diseases. For seven CAG repeat expansion diseases (Huntington's disease (HD) and certain spinocerebellar ataxias), modification of age of onset is linked to variants of specific DNA repair proteins. FAN1 variants are the strongest modifiers. Non-coding disease-delaying FAN1 variants and coding disease-hastening variants (p.R507H and p.R377W) are known, where the former may lead to increased FAN1 levels and the latter have unknown effects upon FAN1 functions. Current thoughts are that ongoing repeat expansions in disease-vulnerable tissues, as individuals age, promote disease onset. Fan1 is required to suppress against high levels of ongoing somatic CAG and CGG repeat expansions in tissues of HD and FMR1 transgenic mice respectively, in addition to participating in DNA interstrand crosslink repair. FAN1 is also a modifier of autism, schizophrenia, and epilepsy. Coupled with the association of these diseases with repeat expansions, this suggests a common mechanism, by which FAN1 modifies repeat diseases. Yet how any of the FAN1 variants modify disease is unknown. Here, we review FAN1 variants, associated clinical effects, protein structure, and the enzyme's attributed functional roles. We highlight how variants may alter its activities in DNA damage response and/or repeat instability. A thorough awareness of the FAN1 gene and FAN1 protein functions will reveal if and how it may be targeted for clinical benefit. [ABSTRACT FROM AUTHOR]

Leishmaniasis and trypanosomiasis are largely neglected diseases prevailing in tropical and subtropical conditions. These are an arthropod-borne zoonosis that affects humans and some animals and is caused by infection with protozoan of the genera Leishmania and Trypanosoma , respectively. These parasites present high genomic plasticity and are able to adapt themselves to adverse conditions like the attack of host cells or toxicity induced by drug exposure. Different mechanisms allow these adapting responses induced by stress, such as mutation, chromosomal rearrangements, establishment of mosaic ploidies, and gene expansion. Here we describe how a subset of genes encoding for DNA polymerases implied in repairing/translesion (TLS) synthesis are duplicated in some pathogenic species of the Trypanosomatida order and a free-living species from the Bodonida order. These enzymes are both able to repair DNA, but are also error-prone under certain situations. We discuss about the possibility that these enzymes can act as a source of genomic variation promoting adaptation in trypanosomatids. [ABSTRACT FROM AUTHOR]

Oligonucleotide/oligosaccharide-binding (OB)-fold proteins play essential roles in the regulation of genome and its correct transformation to the subsequent generation. To maintain the genomic stability, OB-fold proteins are implicated in various cellular processes including DNA replication, DNA repair, cell cycle regulation and maintenance of telomere. The diverse functional spectrums of OB-fold proteins are mainly due to their involvement in protein–DNA and protein–protein complexes. Mutations and consequential structural alteration in the OB-fold proteins often lead to severe diseases. Here, we have investigated the structure, function and mode of action of OB-fold proteins (RPA, BRCA2, DNA ligases and SSBs1/2) in cellular pathways and their relationship with diseases and their possible use in therapeutic intervention. Due to the crucial role of OB-fold proteins in regulating the key physiological process, a detailed structural understanding in the context of underlying mechanism of action and cellular complexity offers a new avenue to target OB-proteins for therapeutic intervention. [ABSTRACT FROM AUTHOR]

The genomes of all living cells are under endogenous and exogenous attacks every day, causing diverse genomic lesions. Most of the lesions can be timely repaired by multiple DNA repair pathways. However, some may persist during S‐phase, block DNA replication, and challenge genome integrity. Eukaryotic cells have evolved DNA damage tolerance (DDT) to mitigate the lethal effects of arrested DNA replication without prior removal of the offending DNA damage. As one important mode of DDT, translesion DNA synthesis (TLS) utilizes multiple low‐fidelity DNA polymerases to incorporate nucleotides opposite DNA lesions to maintain genome integrity. Three different mechanisms have been proposed to regulate the polymerase switching between high‐fidelity DNA polymerases in the replicative machinery and one or more specialized enzymes. Additionally, it is known that proliferating cell nuclear antigen (PCNA) mono‐ubiquitination is essential for optimal TLS. Given its error‐prone property, TLS is closely associated with spontaneous and drug‐induced mutations in cells, which can potentially lead to tumorigenesis and chemotherapy resistance. Therefore, TLS process must be tightly modulated to avoid unwanted mutagenesis. In this review, we will focus on polymerase switching and PCNA mono‐ubiquitination, the two key events in TLS pathway in mammalian cells, and summarize current understandings of regulation of TLS process at the levels of protein–protein interactions, post‐translational modifications as well as transcription and noncoding RNAs. Environ. Mol. Mutagen. 61:680–692, 2020. © 2020 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) production is one of the earliest responses when plants percept pathogens and acts as antimicrobials to block pathogen entry. However, whether and how pathogens tolerate ROS stress remains elusive. Here, we report the chromatin remodeling in Verticillium dahliae, a soil-borne pathogenic fungus that causes vascular wilts of a wide range of plants, facilitates the DNA damage repair in response to plant ROS stress. We identified VdDpb4, encoding a histone-fold protein of the ISW2 chromatin remodeling complex in V. dahliae, is a virulence gene. The reduced virulence in wild type Arabidopsis plants arising from VdDpb4 deletion was impaired in the rbohd mutant plants that did not produce ROS. Further characterization of VdDpb4 and its interacting protein, VdIsw2, an ATP-dependent chromatin-remodeling factor, we show that while the depletion of VdIsw2 led to the decondensing of chromatin, the depletion of VdDpb4 resulted in a more compact chromatin structure and affected the VdIsw2-dependent transcriptional effect on gene expression, including genes involved in DNA damage repair. A knockout mutant of either VdDpb4 or VdIsw2 reduced the efficiency of DNA repair in the presence of DNA-damaging agents and virulence during plant infection. Together, our data demonstrate that VdDpb4 and VdIsw2 play roles in maintaining chromatin structure for positioning nucleosomes and transcription regulation, including genes involved in DNA repair in response to ROS stress during development and plant infection. Author summary: ROS production is one of the earliest responses after the perception of pathogen-associated molecular patterns by plant transmembrane immune receptors, and dependent on the respiratory burst oxidase homolog (RBOH). ROS cause DNA oxidative damage and acts as antimicrobials to block pathogen entry. In this study, we found that chromatin remodeling components, including VdPdb4 and its interacting protein, VdIsw2, are essential for the V. dahliae tolerant in response to ROS stress during development and plant infection. Assays of the accessibility of bulk chromatin suggest that VdDpb4 plays an important role in maintaining a more "open" and accessible chromatin landscape, while VdIsw2 plays an antagonistic role in balancing chromatin structure. Abnormality of nucleosome repositioning by depletion of either protein is harmful to the fungus during DNA repair in response to ROS stress during development and plant infection. We further found that VdDpb4 is required for VdIsw2 to bind to gene promoters for appropriate RNA polymerase II transcription. Taken together, our data demonstrate that VdDpb4 is required for the location of ISW2 on DNA and VdIsw2-dependent transcriptional regulation of gene expression; and provide the first example and essential information for further investigation of chromatin-associated complexes in pathogenic fungi. [ABSTRACT FROM AUTHOR]

The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models. [ABSTRACT FROM AUTHOR]

DNA polymerases are vital for the synthesis of new DNA strands. Since the discovery of DNA polymerase I in Escherichia coli, a diverse library of mammalian DNA polymerases involved in DNA replication, DNA repair, antibody generation, and cell checkpoint signaling has emerged. While the unique functions of these DNA polymerases are differentiated by their association with accessory factors and/or the presence of distinctive catalytic domains, atomic resolution structures of DNA polymerases in complex with their DNA substrates have revealed mechanistic subtleties that contribute to their specialization. In this review, the structure and function of all 15 mammalian DNA polymerases from families B, Y, X, and A will be reviewed and discussed with special emphasis on the insights gleaned from recently published atomic resolution structures. [ABSTRACT FROM AUTHOR]

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, and cell growth and survival. Previously, we identified a novel class of small molecules that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, selectively inhibit tumor cell growth, and induce apoptosis. The purpose of this study was to investigate the combinatorial effects of lead compound PCNA-I1S with DNA damaging agents on cell growth, DNA damage, and DNA repair in four lines of human prostate and lung cancer cells. The DNA damage agents used in the study include ionizing radiation source cesium-137 (Cs-137), chemotherapy drug cisplatin (cisPt), ultraviolet-C (UV-C), and oxidative compound H2O2. DNA damage was assessed using immunofluorescent staining of γH2AX and the Comet assay. The homologous recombination repair (HRR) was determined using a plasmid-based HRR reporter assay and the nucleotide excision repair (NER) was indirectly examined by the removal of UV-induced cyclobutane pyrimidine dimers (CPD). We found that PCNA-I1S inhibited cell growth in a dose-dependent manner and significantly enhanced the cell growth inhibition induced by pretreatment with DNA damaging agents Cs-137 irradiation, UV-C, and cisPt. However, the additive growth inhibitory effects were not observed in cells pre-treated with PCNA-I1S, followed by treatment with cisPt. H2O2 enhanced the level of chromatin-bound PCNA in quiescent cells, which was attenuated by PCNA-I1S. DNA damage was induced in cells treated with either PCNA-I1S or cisPt alone and was significantly elevated in cells exposed to the combination of PCNA-I1S and cisPt. Finally, PCNA-I1S attenuated repair of DNA double strand breaks (DSBs) by HRR and the removal of CPD by NER. These data suggest that targeting PCNA with PCNA-I1S may provide a novel approach for enhancing the efficacy of chemotherapy and radiation therapy in treatment of human prostate and lung cancer. [ABSTRACT FROM AUTHOR]