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Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (31 December 2021) | Viewed by 35485

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Dr. Ennio Prosperi

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Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza”, Consiglio Nazionale delle Ricerche, Genome Stability Group, c/o Department Biology and Biotechnology, Via Ferrata 9, 27100 Pavia, Italy
Interests: DNA damage response (DDR); DNA repair and cell cycle checkpoints; p21(CDKN1A)/PCNA interaction; nucleotide excision repair/base excision repair; post-translational modifications of DNA repair factors; acetylation deficiency and human disease

Dr. Ornella Cazzalini

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Immunology and Pathology Unit, Department of Molecular Medicine, University of Pavia, Via Ferrata 9, 27100 Pavia, Italy
Interests: Cancer Biology; Experimental Medicine

Special Issue Information

Dear Colleagues,

In recent years, knowledge on the cellular and molecular mechanisms underlying the maintenance of genome integrity has increased significantly through the identification of the signaling pathways and main players in the DNA damage response. Thanks to these advancements, crucial aspects of this important cell defense mechanism are currently being exploited, and new strategies to control abnormal cell proliferation and tumor formation are being developed. However, refined definitions of the processes involved in the DNA damage response, especially regarding their regulation, are needed to further this knowledge and identify new points of intervention. In particular, protein–protein interactions, as well as protein post-translation modifications are important components in unravelling the mechanisms responsible for the maintenance of genome stability. This Special Issue will focus on the latest research in the essential processes of DNA replication, repair, and transcription with the aim of advancing the knowledge of the roles of these molecular mechanisms in the DNA damage response.

Dr. Ornella Cazzalini
Dr. Ennio Prosperi
Guest Editors

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Keywords

DNA damage and repair
DNA replication stress
transcription stress
genome stability
protein–protein interactions
post-translational modifications
phosphorylation, acetylation, ubiquitination, poly(ADP)-ribosylation

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Published Papers (9 papers)

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Research

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28 pages, 5898 KiB

Open AccessArticle

The Chromatin Architectural Protein CTCF Is Critical for Cell Survival upon Irradiation-Induced DNA Damage

by Stefania Mamberti, Maruthi K. Pabba, Alexander Rapp, M. Cristina Cardoso and Michael Scholz

Int. J. Mol. Sci. 2022, 23(7), 3896; https://doi.org/10.3390/ijms23073896 - 31 Mar 2022

Cited by 2 | Viewed by 2883

Abstract

CTCF is a nuclear protein initially discovered for its role in enhancer-promoter insulation. It has been shown to play a role in genome architecture and in fact, its DNA binding sites are enriched at the borders of chromatin domains. Recently, we showed that depletion of CTCF impairs the DNA damage response to ionizing radiation. To investigate the relationship between chromatin domains and DNA damage repair, we present here clonogenic survival assays in different cell lines upon CTCF knockdown and ionizing irradiation. The application of a wide range of ionizing irradiation doses (0–10 Gy) allowed us to investigate the survival response through a biophysical model that accounts for the double-strand breaks’ probability distribution onto chromatin domains. We demonstrate that the radiosensitivity of different cell lines is increased upon lowering the amount of the architectural protein. Our model shows that the deficiency in the DNA repair ability is related to the changes in the size of chromatin domains that occur when different amounts of CTCF are present in the nucleus. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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15 pages, 2532 KiB

Open AccessArticle

Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine

by Nadine Müller, Eveliina Ponkkonen, Thomas Carell and Andriy Khobta

Int. J. Mol. Sci. 2021, 22(20), 11025; https://doi.org/10.3390/ijms222011025 - 13 Oct 2021

Cited by 2 | Viewed by 2847

Abstract

Stepwise oxidation of the epigenetic mark 5-methylcytosine and base excision repair (BER) of the resulting 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) may provide a mechanism for reactivation of epigenetically silenced genes; however, the functions of 5-fC and 5-caC at defined gene elements are scarcely explored. We analyzed the expression of reporter constructs containing either 2′-deoxy-(5-fC/5-caC) or their BER-resistant 2′-fluorinated analogs, asymmetrically incorporated into CG-dinucleotide of the GC box cis-element (5′-TGGGCGGAGC) upstream from the RNA polymerase II core promoter. In the absence of BER, 5-caC caused a strong inhibition of the promoter activity, whereas 5-fC had almost no effect, similar to 5-methylcytosine or 5-hydroxymethylcytosine. BER of 5-caC caused a transient but significant promoter reactivation, succeeded by silencing during the following hours. Both responses strictly required thymine DNA glycosylase (TDG); however, the silencing phase additionally demanded a 5′-endonuclease (likely APE1) activity and was also induced by 5-fC or an apurinic/apyrimidinic site. We propose that 5-caC may act as a repressory mark to prevent premature activation of promoters undergoing the final stages of DNA demethylation, when the symmetric CpG methylation has already been lost. Remarkably, the downstream promoter activation or repression responses are regulated by two separate BER steps, where TDG and APE1 act as potential switches. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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14 pages, 2245 KiB

Open AccessArticle

Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG

by Claudia Scalera, Giulio Ticli, Ilaria Dutto, Ornella Cazzalini, Lucia A. Stivala and Ennio Prosperi

Int. J. Mol. Sci. 2021, 22(12), 6589; https://doi.org/10.3390/ijms22126589 - 19 Jun 2021

Cited by 5 | Viewed by 3397

Abstract

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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20 pages, 5606 KiB

Open AccessArticle

O-GlcNAcylation Affects the Pathway Choice of DNA Double-Strand Break Repair

by Sera Averbek, Burkhard Jakob, Marco Durante and Nicole B. Averbeck

Int. J. Mol. Sci. 2021, 22(11), 5715; https://doi.org/10.3390/ijms22115715 - 27 May 2021

Cited by 10 | Viewed by 3143

Abstract

Exposing cells to DNA damaging agents, such as ionizing radiation (IR) or cytotoxic chemicals, can cause DNA double-strand breaks (DSBs), which are crucial to repair to maintain genetic integrity. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification (PTM), which has been reported to be involved in the DNA damage response (DDR) and chromatin remodeling. Here, we investigated the impact of O-GlcNAcylation on the DDR, DSB repair and chromatin status in more detail. We also applied charged particle irradiation to analyze differences of O-GlcNAcylation and its impact on DSB repair in respect of spatial dose deposition and radiation quality. Various techniques were used, such as the γH2AX foci assay, live cell microscopy and Fluorescence Lifetime Microscopy (FLIM) to detect DSB rejoining, protein accumulation and chromatin states after treating the cells with O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) inhibitors. We confirmed that O-GlcNAcylation of MDC1 is increased upon irradiation and identified additional repair factors related to Homologous Recombination (HR), CtIP and BRCA1, which were increasingly O-GlcNAcyated upon irradiation. This is consistent with our findings that the function of HR is affected by OGT inhibition. Besides, we found that OGT and OGA activity modulate chromatin compaction states, providing a potential additional level of DNA-repair regulation. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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20 pages, 3559 KiB

Open AccessArticle

Functional Roles of PARP2 in Assembling Protein–Protein Complexes Involved in Base Excision DNA Repair

by Inna Vasil’eva, Nina Moor, Rashid Anarbaev, Mikhail Kutuzov and Olga Lavrik

Int. J. Mol. Sci. 2021, 22(9), 4679; https://doi.org/10.3390/ijms22094679 - 28 Apr 2021

Cited by 15 | Viewed by 2876

Abstract

Poly(ADP-ribose) polymerase 2 (PARP2) participates in base excision repair (BER) alongside PARP1, but its functions are still under study. Here, we characterize binding affinities of PARP2 for other BER proteins (PARP1, APE1, Polβ, and XRCC1) and oligomerization states of the homo- and hetero-associated complexes using fluorescence-based and light scattering techniques. To compare PARP2 and PARP1 in the efficiency of PAR synthesis, in the absence and presence of protein partners, the size of PARP2 PARylated in various reaction conditions was measured. Unlike PARP1, PARP2 forms more dynamic complexes with common protein partners, and their stability is effectively modulated by DNA intermediates. Apparent binding affinity constants determined for homo- and hetero-oligomerized PARP1 and PARP2 provide evidence that the major form of PARP2 at excessive PARP1 level is their heterocomplex. Autoregulation of PAR elongation at high PARP and NAD⁺ concentrations is stronger for PARP2 than for PARP1, and the activity of PARP2 is more effectively inhibited by XRCC1. Moreover, the activity of both PARP1 and PARP2 is suppressed upon their heteroPARylation. Taken together, our findings suggest that PARP2 can function differently in BER, promoting XRCC1-dependent repair (similarly to PARP1) or an alternative XRCC1-independent mechanism via hetero-oligomerization with PARP1. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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15 pages, 2903 KiB

Open AccessArticle

NEIL1 and NEIL2 Are Recruited as Potential Backup for OGG1 upon OGG1 Depletion or Inhibition by TH5487

by Bishoy M. F. Hanna, Maurice Michel, Thomas Helleday and Oliver Mortusewicz

Int. J. Mol. Sci. 2021, 22(9), 4542; https://doi.org/10.3390/ijms22094542 - 27 Apr 2021

Cited by 16 | Viewed by 3820

Abstract

DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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Review

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20 pages, 1079 KiB

Open AccessReview

Revisiting the Function of p21^CDKN1A in DNA Repair: The Influence of Protein Interactions and Stability

by Giulio Ticli, Ornella Cazzalini, Lucia A. Stivala and Ennio Prosperi

Int. J. Mol. Sci. 2022, 23(13), 7058; https://doi.org/10.3390/ijms23137058 - 24 Jun 2022

Cited by 38 | Viewed by 4252

Abstract

The p21^CDKN1A 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. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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15 pages, 1463 KiB

Open AccessReview

R-Loop-Associated Genomic Instability and Implication of WRN and WRNIP1

by Veronica Marabitti, Pasquale Valenzisi, Giorgia Lillo, Eva Malacaria, Valentina Palermo, Pietro Pichierri and Annapaola Franchitto

Int. J. Mol. Sci. 2022, 23(3), 1547; https://doi.org/10.3390/ijms23031547 - 28 Jan 2022

Cited by 14 | Viewed by 6141

Abstract

Maintenance of genome stability is crucial for cell survival and relies on accurate DNA replication. However, replication fork progression is under constant attack from different exogenous and endogenous factors that can give rise to replication stress, a source of genomic instability and a notable hallmark of pre-cancerous and cancerous cells. Notably, one of the major natural threats for DNA replication is transcription. Encounters or conflicts between replication and transcription are unavoidable, as they compete for the same DNA template, so that collisions occur quite frequently. The main harmful transcription-associated structures are R-loops. These are DNA structures consisting of a DNA–RNA hybrid and a displaced single-stranded DNA, which play important physiological roles. However, if their homeostasis is altered, they become a potent source of replication stress and genome instability giving rise to several human diseases, including cancer. To combat the deleterious consequences of pathological R-loop persistence, cells have evolved multiple mechanisms, and an ever growing number of replication fork protection factors have been implicated in preventing/removing these harmful structures; however, many others are perhaps still unknown. In this review, we report the current knowledge on how aberrant R-loops affect genome integrity and how they are handled, and we discuss our recent findings on the role played by two fork protection factors, the Werner syndrome protein (WRN) and the Werner helicase-interacting protein 1 (WRNIP1) in response to R-loop-induced genome instability. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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25 pages, 1803 KiB

Open AccessReview

RIF1 Links Replication Timing with Fork Reactivation and DNA Double-Strand Break Repair

by Janusz Blasiak, Joanna Szczepańska, Anna Sobczuk, Michal Fila and Elzbieta Pawlowska

Int. J. Mol. Sci. 2021, 22(21), 11440; https://doi.org/10.3390/ijms222111440 - 23 Oct 2021

Cited by 5 | Viewed by 4602

Abstract

Replication timing (RT) is a cellular program to coordinate initiation of DNA replication in all origins within the genome. RIF1 (replication timing regulatory factor 1) is a master regulator of RT in human cells. This role of RIF1 is associated with binding G4-quadruplexes and changes in 3D chromatin that may suppress origin activation over a long distance. Many effects of RIF1 in fork reactivation and DNA double-strand (DSB) repair (DSBR) are underlined by its interaction with TP53BP1 (tumor protein p53 binding protein). In G1, RIF1 acts antagonistically to BRCA1 (BRCA1 DNA repair associated), suppressing end resection and homologous recombination repair (HRR) and promoting non-homologous end joining (NHEJ), contributing to DSBR pathway choice. RIF1 is an important element of intra-S-checkpoints to recover damaged replication fork with the involvement of HRR. High-resolution microscopic studies show that RIF1 cooperates with TP53BP1 to preserve 3D structure and epigenetic markers of genomic loci disrupted by DSBs. Apart from TP53BP1, RIF1 interact with many other proteins, including proteins involved in DNA damage response, cell cycle regulation, and chromatin remodeling. As impaired RT, DSBR and fork reactivation are associated with genomic instability, a hallmark of malignant transformation, RIF1 has a diagnostic, prognostic, and therapeutic potential in cancer. Further studies may reveal other aspects of common regulation of RT, DSBR, and fork reactivation by RIF1. Full article

(This article belongs to the Special Issue Molecular Mechanisms Safeguarding Genome Integrity in DNA Replication, Repair and Transcription)

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