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Molecular Mechanism of DNA Replication and Repair

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 (28 February 2023) | Viewed by 17353

Special Issue Editors

Special Issue Information

Dear Colleagues,

During cell division, the replisome machinery is capable of quickly and accurately copying billions of DNA bases in order to maintain genome integrity and prevent diseases. Nevertheless, it can make errors, particularly when the DNA is damaged. dsDNA is a stable, chemically inert molecule continuously subjected to a variety of exogenous and endogenous insults throughout the cell cycle. Unrepaired or misrepaired DNA lesions may cause mutations or chromosomal damage and, ultimately, abnormalities, including oncogenic transformation. For the maintenance of genomic stability, the organisms have developed signal pathways that give rise to a DNA damage response (DDR). This mechanism is characterized by its capability to tolerate DNA damage and structural impediments during DNA synthesis; thus, it replicates fork progression and stability in the presence of blocking structures or DNA lesions. Malfunctioning DDR can cause fork arrest and eventually fork collapse, leading to the formation of DNA double strand breaks.

This Special Issue of IJMS aims to expand our current understanding on molecular and cellular mechanisms of DNA replication and repair. We want to offer a platform for high-quality publications on various sides of DNA replication and repair research. Bringing together different aspects in one issue, hopefully, will trigger findings relevant to the development of new therapies in human diseases.

Dr. Mariarita De Felice
Dr. Mariarosaria De Falco
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

  • genome stability
  • DNA repair
  • DNA replication
  • DDR

Published Papers (9 papers)

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Research

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20 pages, 4503 KiB  
Article
pilE G-Quadruplex Is Recognized and Preferentially Bound but Not Processed by the MutL Endonuclease from Neisseria gonorrhoeae Mismatch Repair Pathway
Int. J. Mol. Sci. 2023, 24(7), 6167; https://doi.org/10.3390/ijms24076167 - 24 Mar 2023
Viewed by 1114
Abstract
The human pathogen Neisseria gonorrhoeae uses a homologous recombination to undergo antigenic variation and avoid an immune response. The surface protein pilin (PilE) is one of the targets for antigenic variation that can be regulated by N. gonorrhoeae mismatch repair (MMR) and a [...] Read more.
The human pathogen Neisseria gonorrhoeae uses a homologous recombination to undergo antigenic variation and avoid an immune response. The surface protein pilin (PilE) is one of the targets for antigenic variation that can be regulated by N. gonorrhoeae mismatch repair (MMR) and a G-quadruplex (G4) located upstream of the pilE promoter. Using bioinformatics tools, we found a correlation between pilE variability and deletion of DNA regions encoding ngMutS or ngMutL proteins, the main participants in N. gonorrhoeae methyl-independent MMR. To understand whether the G4 structure could affect the ngMutL-mediated regulation of pilin antigenic variation, we designed several synthetic pilE G4-containing oligonucleotides, differing in length, and related DNA duplexes. Using CD measurements and biochemical approaches, we have showed that (i) ngMutL preferentially binds to pilE G4 compared to DNA duplex, although the latter is a cognate substrate for ngMutL endonuclease, (ii) protein binding affinity decreases with shortening of quadruplex-containing and duplex ligands, (iii) the G4 structure inhibits ngMutL-induced DNA nicking and modulates cleavage positions; the enzyme does not cleave DNA within G4, but is able to bypass this noncanonical structure. Thus, pilE G4 may regulate the efficiency of pilin antigenic variation by quadruplex binding to ngMutL and suppression of homologous recombination. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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16 pages, 4675 KiB  
Article
Human Polβ Natural Polymorphic Variants G118V and R149I Affects Substate Binding and Catalysis
Int. J. Mol. Sci. 2023, 24(6), 5892; https://doi.org/10.3390/ijms24065892 - 20 Mar 2023
Cited by 3 | Viewed by 1061
Abstract
DNA polymerase β (Polβ) expression is essential for the cell’s response to DNA damage that occurs during natural cellular processes. Polβ is considered the main reparative DNA polymerase, whose role is to fill the DNA gaps arising in the base excision repair pathway. [...] Read more.
DNA polymerase β (Polβ) expression is essential for the cell’s response to DNA damage that occurs during natural cellular processes. Polβ is considered the main reparative DNA polymerase, whose role is to fill the DNA gaps arising in the base excision repair pathway. Mutations in Polβ can lead to cancer, neurodegenerative diseases, or premature aging. Many single-nucleotide polymorphisms have been identified in the POLB gene, but the consequences of these polymorphisms are not always clear. It is known that some polymorphic variants in the Polβ sequence reduce the efficiency of DNA repair, thereby raising the frequency of mutations in the genome. In the current work, we studied two polymorphic variants (G118V and R149I separately) of human Polβ that affect its DNA-binding region. It was found that each amino acid substitution alters Polβ’s affinity for gapped DNA. Each polymorphic variant also weakens its binding affinity for dATP. The G118V variant was found to greatly affect Polβ’s ability to fill gapped DNA and slowed the catalytic rate as compared to the wild-type enzyme. Thus, these polymorphic variants seem to decrease the ability of Polβ to maintain base excision repair efficiency. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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14 pages, 1556 KiB  
Article
Regulation of ssb Gene Expression in Escherichia coli
Int. J. Mol. Sci. 2022, 23(18), 10917; https://doi.org/10.3390/ijms231810917 - 18 Sep 2022
Cited by 3 | Viewed by 2364
Abstract
Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by [...] Read more.
Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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14 pages, 1470 KiB  
Article
Conformational Rearrangements Regulating the DNA Repair Protein APE1
Int. J. Mol. Sci. 2022, 23(14), 8015; https://doi.org/10.3390/ijms23148015 - 20 Jul 2022
Cited by 1 | Viewed by 1606
Abstract
Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for [...] Read more.
Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for the correction by subsequent enzymes of the BER machinery. In spite of a wealth of information on APE1 structure and activity, its regulation mechanism still remains to be understood. Human APE1 consists of a globular catalytic domain preceded by a flexible N-terminal extension, which might be involved in the interaction with DNA. Moreover, the binding of the nuclear chaperone nucleophosmin (NPM1) to this region has been reported to impact APE1 catalysis. To evaluate intra- and inter-molecular conformational rearrangements upon DNA binding, incision, and interaction with NPM1, we used Förster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to molecular distances. Our results suggest that the N-terminus approaches the DNA at the downstream side of the abasic site and enables the building of a predictive model of the full-length APE1/DNA complex. Furthermore, the spatial configuration of the N-terminal tail is sensitive to NPM1, which could be related to the regulation of APE1. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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14 pages, 7903 KiB  
Article
A Homozygous Loss-of-Function Mutation in MSH5 Abolishes MutSγ Axial Loading and Causes Meiotic Arrest in NOA-Affected Individuals
Int. J. Mol. Sci. 2022, 23(12), 6522; https://doi.org/10.3390/ijms23126522 - 10 Jun 2022
Cited by 3 | Viewed by 1868
Abstract
Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was [...] Read more.
Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was identified from two idiopathic NOA patients in the consanguineous family. This mutation led to the degradation of MSH5 mRNA and abolished chromosome axial localization of MutSγ in spermatocytes from the affected males. Chromosomal spreading analysis of the patient’s meiotic prophase I revealed that the meiosis progression was arrested at a zygotene-like stage with extensive failure of homologous synapsis and DSB repair. Therefore, our study demonstrates that the MSH5 c.1126del could cause meiotic recombination failure and lead to human infertility, improving the genetic diagnosis of NOA clinically. Furthermore, the study of human spermatocytes elucidates the meiosis defects caused by MSH5 variant, and reveals a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not previously been well-described. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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16 pages, 1956 KiB  
Article
Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals
Int. J. Mol. Sci. 2022, 23(7), 3973; https://doi.org/10.3390/ijms23073973 - 02 Apr 2022
Viewed by 2646
Abstract
Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. [...] Read more.
Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (αLA-LPH Tg goat, Akr1A1eGFP/eGFP Tg mouse, and NFκB-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (αLA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5′ homologous arm inverted repeat donor plasmid (5′IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3′ inverted terminal repeat/inverted repeat donor plasmid (3′ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3′-overhang integration into the host genome via homologous recombination machinery. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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15 pages, 3190 KiB  
Article
Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes
Int. J. Mol. Sci. 2022, 23(5), 2869; https://doi.org/10.3390/ijms23052869 - 06 Mar 2022
Cited by 3 | Viewed by 1678
Abstract
Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed [...] Read more.
Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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16 pages, 2911 KiB  
Article
The Finely Coordinated Action of SSB and NurA/HerA Complex Strictly Regulates the DNA End Resection Process in Saccharolobus solfataricus
Int. J. Mol. Sci. 2022, 23(5), 2582; https://doi.org/10.3390/ijms23052582 - 26 Feb 2022
Viewed by 1672
Abstract
Generation of the 3′ overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved [...] Read more.
Generation of the 3′ overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3′ single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus. We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when SSB is present in the reaction. In accordance with what has been observed, we have shown that HerA helicase activity is not stimulated by SSB. These data suggest that, in archaea, the DNA end resection process is governed by the strictly combined action of NurA, HerA and SSB. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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Review

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22 pages, 2419 KiB  
Review
Noncatalytic Domains in DNA Glycosylases
Int. J. Mol. Sci. 2022, 23(13), 7286; https://doi.org/10.3390/ijms23137286 - 30 Jun 2022
Viewed by 1632
Abstract
Many proteins consist of two or more structural domains: separate parts that have a defined structure and function. For example, in enzymes, the catalytic activity is often localized in a core fragment, while other domains or disordered parts of the same protein participate [...] Read more.
Many proteins consist of two or more structural domains: separate parts that have a defined structure and function. For example, in enzymes, the catalytic activity is often localized in a core fragment, while other domains or disordered parts of the same protein participate in a number of regulatory processes. This situation is often observed in many DNA glycosylases, the proteins that remove damaged nucleobases thus initiating base excision DNA repair. This review covers the present knowledge about the functions and evolution of such noncatalytic parts in DNA glycosylases, mostly concerned with the human enzymes but also considering some unique members of this group coming from plants and prokaryotes. Full article
(This article belongs to the Special Issue Molecular Mechanism of DNA Replication and Repair)
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