SciCombinator

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Journal: DNA repair

27

Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell.

Concepts: DNA, Protein, Gene, Genetics, Cell nucleus, Bacteria, Organism, DNA replication

25

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism.

Concepts: DNA, Protein, Metabolism, Adenosine triphosphate, DNA repair, DNA replication, Molecular motor, Helicase

23

Acylpeptide hydrolase (APEH) deacetylates N-alpha-acetylated peptides and selectively degrades oxidised proteins, but the biochemical pathways that are regulated by this protease are unknown. Here, we identify APEH as a component of the cellular response to DNA damage. Although APEH is primarily localised in the cytoplasm, we show that a sub-fraction of this enzyme is sequestered at sites of nuclear damage following UVA irradiation or following oxidative stress. We show that localization of APEH at sites of nuclear damage is mediated by direct interaction with XRCC1, a scaffold protein that accelerates the repair of DNA single-strand breaks. We show that APEH interacts with the amino-terminal domain of XRCC1, and that APEH facilitates both single-strand break repair and cell survival following exposure to H2O2 in human cells. These data identify APEH as a novel proteolytic component of the DNA damage response.

Concepts: DNA, Protein, Gene, Cell nucleus, Cell, Metabolism, Adenosine triphosphate, Enzyme

5

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.

Concepts: DNA, Signal transduction, Adenosine triphosphate, DNA repair, Phosphorylation, DNA replication, Non-homologous end joining, DNA-PKcs

4

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication.

Concepts: DNA, Protein, Gene, Adenosine triphosphate, Enzyme, DNA repair, Kinase, DNA replication

1

Upon DNA binding the poly(ADP-ribose) polymerase family of enzymes (PARPs) add multiple ADP-ribose subunits to themselves and other acceptor proteins. Inhibitors of PARPs have become an exciting and real prospect for monotherapy and as sensitizers to ionising radiation (IR). The action of PARPs are reversed by poly(ADP-ribose) glycohydrolase (PARG). Until recently studies of PARG have been limited by the lack of an inhibitor. Here, a first in class, specific, and cell permeable PARG inhibitor, PDD00017273, is shown to radiosensitize. Further, PDD00017273 is compared with the PARP1/2/3 inhibitor olaparib. Both olaparib and PDD00017273 altered the repair of IR-induced DNA damage, resulting in delayed resolution of RAD51 foci compared with control cells. However, only PARG inhibition induced a rapid increase in IR-induced activation of PRKDC (DNA-PK) and perturbed mitotic progression. This suggests that PARG has additional functions in the cell compared with inhibition of PARP1/2/3, likely via reversal of tankyrase activity and/or that inhibiting the removal of poly(ADP-ribose) (PAR) has a different consequence to inhibiting PAR addition. Overall, our data are consistent with previous genetic findings, reveal new insights into the function of PAR metabolism following IR and demonstrate for the first time the therapeutic potential of PARG inhibitors as radiosensitizing agents.

Concepts: DNA, Protein, Gene, Bacteria, Radiation therapy, Enzyme inhibitor, Inhibitor, Xanthine oxidase inhibitor

1

Maintenance of a genome requires DNA repair integrated with chromatin remodeling. We have analyzed six transcriptome data sets and one data set on translational regulation of known DNA repair and remodeling genes in synchronized human cells. These data are available through our new database: www.dnarepairgenes.com. Genes that have similar transcription profiles in at least two of our data sets generally agree well with known protein profiles. In brief, long patch base excision repair (BER) is enriched for S phase genes, whereas short patch BER uses genes essentially equally expressed in all cell cycle phases. Furthermore, most genes related to DNA mismatch repair, Fanconi anemia and homologous recombination have their highest expression in the S phase. In contrast, genes specific for direct repair, nucleotide excision repair, as well as non-homologous end joining do not show cell cycle-related expression. Cell cycle regulated chromatin remodeling genes were most frequently confined to G1/S and S. These include e.g. genes for chromatin assembly factor 1 (CAF-1) major subunits CHAF1A and CHAF1B; the putative helicases HELLS and ATAD2 that both co-activate E2F transcription factors central in G1/S-transition and recruit DNA repair and chromatin-modifying proteins and DNA double strand break repair proteins; and RAD54L and RAD54B involved in double strand break repair. TOP2A was consistently most highly expressed in G2, but also expressed in late S phase, supporting a role in regulating entry into mitosis. Translational regulation complements transcriptional regulation and appears to be a relatively common cell cycle regulatory mechanism for DNA repair genes. Our results identify cell cycle phases in which different pathways have highest activity, and demonstrate that periodically expressed genes in a pathway are frequently co-expressed. Furthermore, the data suggest that S phase expression and over-expression of some multifunctional chromatin remodeling proteins may set up feedback loops driving cancer cell proliferation.

Concepts: DNA, Protein, Gene, Genetics, Cell nucleus, Gene expression, DNA repair, Cell cycle

0

The DNA-damaging agent 5-fluorouracil represents the most commonly used chemotherapeutic drug for colorectal cancer patients. DNA lesions associated with 5-fluorouracil therapy are primarily repaired by base excision repair (BER) and mismatch repair (MMR) pathways. Published evidence suggests that the individual DNA repair capacity (DRC) may affect a patient’s prognosis and response to chemotherapy. With this in mind, we designed a prospective study of which the main aim was to investigate BER-DRC in relation to 5-fluorouracil response as potential predictive and/or prognostic biomarker. BER-DRC was supplemented by a microsatellite instability (MSI) analysis which represents an indirect marker of MMR activity in the tumor. All parameters were measured in paired samples of tumor tissue and non-malignant adjacent mucosa of 123 incident colon cancer patients. Our results indicate that BER-DRC in non-malignant adjacent mucosa was positively associated with overall survival (P = 0.007) and relapse-free survival (P = 0.04). Additionally, in multivariate analysis, good therapy responders in TNM stage II and III with an elevated BER-DRC in mucosa exhibited better overall survival. Moreover, the overall survival of these patients was even better in the presence of a decreased BER-DRC in tumor tissue. The ratio of BER-DRC in tumor tissue over BER-DRC in mucosa positively correlated with advanced tumor stage (P = 0.003). With respect to MSI, we observed that MSI-high tumors were mostly localized in proximal colon; however, in our cohort, the MSI status affected neither patients' prognosis nor survival. In summary, the results of the present study suggest that the level of BER-DRC is associated with patients' survival. BER-DRC represents a potential prognostic biomarker, applicable for prediction of therapy response and useful for individual approach to patients.

0

Stalled RNA polymerases (RNAPs) pose an obstacle for the replicating complexes, which could lead to transcription-replication conflicts and result in genetic instability. Stalled RNAPs and DNA lesions blocking RNAP elongation are removed by transcription-coupled repair (TCR), the process which in bacteria is mediated by TCR factor Mfd and helicase UvrD. Although the mechanism of TCR has been extensively studied, its role in mutagenesis is still obscure. In the current study we have investigated the role of Mfd and UvrD in mutational processes in soil bacterium Pseudomonas putida. Our results revealed that UvrD helicase is essential to prevent the emergence of mutations, as the loss of uvrD resulted in elevated mutant frequency both in exponential- and stationary-phase bacterial cultures. UvrD was also found to be necessary to survive DNA damage, but NER or MMR pathways are not completely abolished in UvrD-deficient P. putida. Mfd-deficiency had a moderate impact on surviving DNA damage and did not influence the frequency of mutations occurred in exponentially growing bacteria. However, the absence of Mfd caused approximately a two-fold decline in stationary-phase mutant frequency compared to the P. putida wild-type strain and suppressed the elevated mutant frequency observed in the ΔuvrD strain. Remarkably, the Mfd-deficient strain also formed less UV-induced mutants. These results suggest that in P. putida the Mfd-mediated TCR could be associated with UV- and stationary-phase mutagenesis.

0

Replication protein A contributes to all major pathways of DNA metabolism and is a target for post-translation modifications, including poly(ADP-ribosyl)ation catalyzed by PARP1. Here we demonstrate that the efficiency of RPA poly(ADP-ribosyl)ation strongly depends on the structure of DNA used for PARP1 activation and on the polarity of RPA binding. Moreover, RPA influences PARP1 activity, and this effect also depends on DNA structure: RPA inhibits PAR synthesis catalyzed by PARP1 in the presence of ssDNA and stimulates it in the presence of a DNA duplex, in particular that containing a nick or a gap. Using fluorescently labeled proteins, we showed their direct interaction and characterized it quantitatively. RPA can accelerate the replacement of poly(ADP-ribosyl)ated PARP1 molecules bound to DNA by the unmodified ones. Thus, our data allow us to suggest that the balance between the affinities of PARP1 and RPA for DNA and the interaction of these proteins with each other are the cornerstone of the modulating effect of RPA on PARP1 activity. This effect might contribute to the regulation of PARP1 activity in various DNA processing mechanisms including DNA replication and repair pathways, where both PARP1 and RPA participate.