bims-numges 2022-02-27 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022‒02‒27
thirty-two papers selected by
Sean Rudd
Karolinska Institutet

Re-Discovery of Pyrimidine Salvage as Target in Cancer Therapy.
Multifunctional role of thymidine phosphorylase in cancer.
RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor.
The inner workings of replisome-dependent control of DNA damage tolerance.
DNA Damage Tolerance Pathways in Human Cells: A Potential Therapeutic Target.
PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy.
Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins.
The Dynamic Behavior of Chromatin in Response to DNA Double-Strand Breaks.
The Role of Natural Polymorphic Variants of DNA Polymerase β in DNA Repair.
DNA Repair in Space and Time: Safeguarding the Genome with the Cohesin Complex.
Cryo-EM structure of translesion DNA synthesis polymerase ζ with a base pair mismatch.
NBS1-CtIP-mediated DNA end resection suppresses cGAS binding to micronuclei.
Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.
Small Cajal body-associated RNA 2 (scaRNA2) regulates DNA repair pathway choice by inhibiting DNA-PK.
RIF1 acts in DNA repair through phosphopeptide recognition of 53BP1.
Regulation of Replication Stress in Alternative Lengthening of Telomeres by Fanconi Anaemia Protein.
Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells using the DNA Fiber Assay.
A Low-Activity Polymorphic Variant of Human NEIL2 DNA Glycosylase.
XRCC1 counteracts PARP poisons, Olaparib and Talazoparib, and a clinical alkylating agent, Temozolomide, by promoting the removal of trapped PARP1 from broken DNA.
The Multiple Roles of Glucose-6-Phosphate Dehydrogenase in Tumorigenesis and Cancer Chemoresistance.
Stalling of Eukaryotic Translesion DNA Polymerases at DNA-Protein Cross-Links.
Everything Comes with a Price: The Toxicity Profile of DNA-Damage Response Targeting Agents.
A mechanism of origin licensing control through autoinhibition of S. cerevisiae ORC·DNA·Cdc6.
RIOX1-demethylated cGAS regulates ionizing radiation-elicited DNA repair.
Precision Medicine for BRCA/PALB2-Mutated Pancreatic Cancer and Emerging Strategies to Improve Therapeutic Responses to PARP Inhibition.
Ubiquitin and SUMO as timers during DNA replication.
p53-NEIL1 co-abnormalities induce genomic instability and promote synthetic lethality with Chk1 inhibition in multiple myeloma having concomitant 17p13(del) and 1q21(gain).
Examination of multiple Trypanosoma cruzi targets in a new drug discovery approach for Chagas disease.
IMPDH dysregulation in disease: a mini review.
Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR.
Protein-lipid interactions of human dihydroorotate dehydrogenase and three mutants associated with Miller syndrome.
Inborn errors of purine and pyrimidine metabolism: A guide to diagnosis.

Cells. 2022 Feb 20. pii: 739. [Epub ahead of print]11(4):

Re-Discovery of Pyrimidine Salvage as Target in Cancer Therapy.

Melanie Walter, Patrick Herr.

  Nucleotides are synthesized through two distinct pathways: de novo synthesis and nucleoside salvage. Whereas the de novo pathway synthesizes nucleotides from amino acids and glucose, the salvage pathway recovers nucleosides or bases formed during DNA or RNA degradation. In contrast to high proliferating non-malignant cells, which are highly dependent on the de novo synthesis, cancer cells can switch to the nucleoside salvage pathways to maintain efficient DNA replication. Pyrimidine de novo synthesis remains the target of interest in cancer therapy and several inhibitors showed promising results in cancer cells and in vivo models. In the 1980s and 1990s, poor responses were however observed in clinical trials with several of the currently existing pyrimidine synthesis inhibitors. To overcome the observed limitations in clinical trials, targeting pyrimidine salvage alone or in combination with pyrimidine de novo inhibitors was suggested. Even though this approach showed initially promising results, it received fresh attention only recently. Here we discuss the re-discovery of targeting pyrimidine salvage pathways for DNA replication alone or in combination with inhibitors of pyrimidine de novo synthesis to overcome limitations of commonly used antimetabolites in various preclinical cancer models and clinical trials. We also highlight newly emerged targets in pyrimidine synthesis as well as pyrimidine salvage as a promising target in immunotherapy.

Keywords:  DNA replication; cancer therapy; nucleotide metabolism; pyrimidine salvage; replication stress

DOI:  https://doi.org/10.3390/cells11040739
Trends Cancer. 2022 Feb 19. pii: S2405-8033(22)00025-5. [Epub ahead of print]

Multifunctional role of thymidine phosphorylase in cancer.

Becka M Warfield, Philip Reigan.

  Thymidine phosphorylase (TP) catalyzes the reversible phosphorolysis of thymidine, maintaining nucleoside homeostasis for DNA repair and replication. In many cancers TP is expressed at high levels and promotes thymidine catabolism, ultimately generating 2-deoxyribose (2dDR) that can support multiple procancer processes, including glycation of proteins, alternative metabolism, extracellular matrix remodeling, and angiogenesis. Therefore, inhibition of TP is an attractive anticancer strategy; however, an alternative approach that exploits the catalytic activity of TP to activate 5-fluorouracil (5-FU) prodrugs has been clinically successful. Here, we review the structure, function, and regulation of TP, its multiple supporting roles in cancer growth and survival. We summarize TP inhibitor and prodrug development and propose TP-targeting strategies that could potentiate the action of current therapies.

Keywords:  DNA repair; Src family kinase; angiogenesis; glycation; inhibitor; metabolism; prodrug; thymidine phosphorylase

DOI:  https://doi.org/10.1016/j.trecan.2022.01.018
Cells. 2022 Feb 12. pii: 643. [Epub ahead of print]11(4):

RNAi Screening Uncovers a Synthetic Sick Interaction between CtIP and the BARD1 Tumor Suppressor.

Hella A Bolck, Sara Przetocka, Roger Meier, Christine von Aesch, Christina Zurfluh, Kay Hänggi, Vincent Spegg, Matthias Altmeyer, Michael Stebler, Simon F Nørrelykke, Peter Horvath, Alessandro A Sartori, Antonio Porro.

  Human CtIP is best known for its role in DNA end resection to initiate DNA double-strand break repair by homologous recombination. Recently, CtIP has also been shown to protect reversed replication forks from nucleolytic degradation upon DNA replication stress. However, still little is known about the DNA damage response (DDR) networks that preserve genome integrity and sustain cell survival in the context of CtIP insufficiency. Here, to reveal such potential buffering relationships, we screened a DDR siRNA library in CtIP-deficient cells to identify candidate genes that induce synthetic sickness/lethality (SSL). Our analyses unveil a negative genetic interaction between CtIP and BARD1, the heterodimeric binding partner of BRCA1. We found that simultaneous disruption of CtIP and BARD1 triggers enhanced apoptosis due to persistent replication stress-induced DNA lesions giving rise to chromosomal abnormalities. Moreover, we observed that the genetic interaction between CtIP and BARD1 occurs independently of the BRCA1-BARD1 complex formation and might be, therefore, therapeutical relevant for the treatment of BRCA-defective tumors.

Keywords:  BARD1; BRCA1; CtIP; DNA damage; replication stress; synthetic lethality

DOI:  https://doi.org/10.3390/cells11040643
Genes Dev. 2022 Feb 01. 36(3-4): 103-105

The inner workings of replisome-dependent control of DNA damage tolerance.

Tianpeng Zhang, Roger A Greenberg.

  Genomic DNA is continuously challenged by endogenous and exogenous sources of damage. The resulting lesions may act as physical blocks to DNA replication, necessitating repair mechanisms to be intrinsically coupled to the DNA replisome machinery. DNA damage tolerance (DDT) is comprised of translesion synthesis (TLS) and template switch (TS) repair processes that allow the replisome to bypass of bulky DNA lesions and complete DNA replication. How the replisome orchestrates which DDT repair mechanism becomes active at replication blocks has remained enigmatic. In this issue of Genes & Development, Dolce and colleagues (pp. 167-179) report that parental histone deposition by replisome components Ctf4 and Dpb3/4 promotes TS while suppressing error-prone TLS. Deletion of Dpb3/4 restored resistance to DNA-damaging agents in ctf4Δ cells at the expense of synergistic increases in mutagenesis due to elevated TLS. These findings illustrate the importance of replisome-directed chromatin maintenance to genome integrity and the response to DNA-damaging anticancer therapeutics.

Keywords:  DNA damage tolerance; Dpb3–Dpb4; Mcm2–Ctf4–Polα; histone deposition; mutagenesis; recombination; replication fork

DOI:  https://doi.org/10.1101/gad.349408.122
Front Oncol. 2021 ;11 822500

DNA Damage Tolerance Pathways in Human Cells: A Potential Therapeutic Target.

Ashlynn Ai Li Ler, Michael P Carty.

  DNA lesions arising from both exogenous and endogenous sources occur frequently in DNA. During DNA replication, the presence of unrepaired DNA damage in the template can arrest replication fork progression, leading to fork collapse, double-strand break formation, and to genome instability. To facilitate completion of replication and prevent the generation of strand breaks, DNA damage tolerance (DDT) pathways play a key role in allowing replication to proceed in the presence of lesions in the template. The two main DDT pathways are translesion synthesis (TLS), which involves the recruitment of specialized TLS polymerases to the site of replication arrest to bypass lesions, and homology-directed damage tolerance, which includes the template switching and fork reversal pathways. With some exceptions, lesion bypass by TLS polymerases is a source of mutagenesis, potentially contributing to the development of cancer. The capacity of TLS polymerases to bypass replication-blocking lesions induced by anti-cancer drugs such as cisplatin can also contribute to tumor chemoresistance. On the other hand, during homology-directed DDT the nascent sister strand is transiently utilised as a template for replication, allowing for error-free lesion bypass. Given the role of DNA damage tolerance pathways in replication, mutagenesis and chemoresistance, a more complete understanding of these pathways can provide avenues for therapeutic exploitation. A number of small molecule inhibitors of TLS polymerase activity have been identified that show synergy with conventional chemotherapeutic agents in killing cancer cells. In this review, we will summarize the major DDT pathways, explore the relationship between damage tolerance and carcinogenesis, and discuss the potential of targeting TLS polymerases as a therapeutic approach.

Keywords:  DNA damage; DNA damage tolerance pathways; DNA replication; TLS inhibitors; cancer therapeutics; translesion synthesis (TLS)

DOI:  https://doi.org/10.3389/fonc.2021.822500
Biomolecules. 2022 Feb 03. pii: 248. [Epub ahead of print]12(2):

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy.

Alberto Díaz-Talavera, Cristina Montero-Conde, Luis Javier Leandro-García, Mercedes Robledo.

  DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with convectional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a translesion synthesis (TLS) DNA primase, elongating primers that PrimPol itself sythesizes, or as TLS DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only uses its TLS abilities to facilitate priming across lesions in cells, thus acting as a TLS DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer.

Keywords:  PrimPol; cancer; genomic instability; polymerase; primase; replication stress; repriming

DOI:  https://doi.org/10.3390/biom12020248
Nat Commun. 2022 02 21. 13(1): 974

Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins.

Namrata Kumar, Arjan F Theil, Vera Roginskaya, Yasmin Ali, Michael Calderon, Simon C Watkins, Ryan P Barnes, Patricia L Opresko, Alex Pines, Hannes Lans, Wim Vermeulen, Bennett Van Houten.

UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions.

DOI: https://doi.org/10.1038/s41467-022-28642-9
Genes (Basel). 2022 Jan 25. pii: 215. [Epub ahead of print]13(2):

The Dynamic Behavior of Chromatin in Response to DNA Double-Strand Breaks.

Fabiola García Fernández, Emmanuelle Fabre.

  The primary functions of the eukaryotic nucleus as a site for the storage, retrieval, and replication of information require a highly dynamic chromatin organization, which can be affected by the presence of DNA damage. In response to double-strand breaks (DSBs), the mobility of chromatin at the break site is severely affected and, to a lesser extent, that of other chromosomes. The how and why of such movement has been widely studied over the last two decades, leading to different mechanistic models and proposed potential roles underlying both local and global mobility. Here, we review the state of the knowledge on current issues affecting chromatin mobility upon DSBs, and highlight its role as a crucial step in the DNA damage response (DDR).

Keywords:  DNA damage response (DDR); chromatin; chromatin dynamics; double strand break (DSB); genome integrity

DOI:  https://doi.org/10.3390/genes13020215
Int J Mol Sci. 2022 Feb 21. pii: 2390. [Epub ahead of print]23(4):

The Role of Natural Polymorphic Variants of DNA Polymerase β in DNA Repair.

Olga A Kladova, Olga S Fedorova, Nikita A Kuznetsov.

  DNA polymerase β (Polβ) is considered the main repair DNA polymerase involved in the base excision repair (BER) pathway, which plays an important part in the repair of damaged DNA bases usually resulting from alkylation or oxidation. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that protein-protein interactions of Polβ with enzymes from the BER pathway increase the efficiency of damaged base repair in DNA. However natural single-nucleotide polymorphisms can lead to a substitution of functionally significant amino acid residues and therefore affect the catalytic activity of the enzyme and the accuracy of Polβ action. Up-to-date databases contain information about more than 8000 SNPs in the gene of Polβ. This review summarizes data on the in silico prediction of the effects of Polβ SNPs on DNA repair efficacy; available data on cancers associated with SNPs of Polβ; and experimentally tested variants of Polβ. Analysis of the literature indicates that amino acid substitutions could be important for the maintenance of the native structure of Polβ and contacts with DNA; others affect the catalytic activity of the enzyme or play a part in the precise and correct attachment of the required nucleotide triphosphate. Moreover, the amino acid substitutions in Polβ can disturb interactions with enzymes involved in BER, while the enzymatic activity of the polymorphic variant may not differ significantly from that of the wild-type enzyme. Therefore, investigation regarding the effect of Polβ natural variants occurring in the human population on enzymatic activity and protein-protein interactions is an urgent scientific task.

Keywords:  DNA polymerase beta; DNA repair; DNA repair coordination; enzymatic activity; protein–protein interaction; single-nucleotide polymorphism

DOI:  https://doi.org/10.3390/ijms23042390
Genes (Basel). 2022 Jan 22. pii: 198. [Epub ahead of print]13(2):

DNA Repair in Space and Time: Safeguarding the Genome with the Cohesin Complex.

Jamie Phipps, Karine Dubrana.

  DNA double-strand breaks (DSBs) are a deleterious form of DNA damage, which must be robustly addressed to ensure genome stability. Defective repair can result in chromosome loss, point mutations, loss of heterozygosity or chromosomal rearrangements, which could lead to oncogenesis or cell death. We explore the requirements for the successful repair of DNA DSBs by non-homologous end joining and homology-directed repair (HDR) mechanisms in relation to genome folding and dynamics. On the occurrence of a DSB, local and global chromatin composition and dynamics, as well as 3D genome organization and break localization within the nuclear space, influence how repair proceeds. The cohesin complex is increasingly implicated as a key regulator of the genome, influencing chromatin composition and dynamics, and crucially genome organization through folding chromosomes by an active loop extrusion mechanism, and maintaining sister chromatid cohesion. Here, we consider how this complex is now emerging as a key player in the DNA damage response, influencing repair pathway choice and efficiency.

Keywords:  DNA repair; NHEJ; chromatin; chromatin dynamics; cohesin; homologous recombination; nuclear organization

DOI:  https://doi.org/10.3390/genes13020198
Nat Commun. 2022 Feb 25. 13(1): 1050

Cryo-EM structure of translesion DNA synthesis polymerase ζ with a base pair mismatch.

Radhika Malik, Robert E Johnson, Louise Prakash, Satya Prakash, Iban Ubarretxena-Belandia, Aneel K Aggarwal.

The B-family multi-subunit DNA polymerase ζ (Polζ) is important for translesion DNA synthesis (TLS) during replication, due to its ability to extend synthesis past nucleotides opposite DNA lesions and mismatched base pairs. We present a cryo-EM structure of Saccharomyces cerevisiae Polζ with an A:C mismatch at the primer terminus. The structure shows how the Polζ active site responds to the mismatched duplex DNA distortion, including the loosening of key protein-DNA interactions and a fingers domain in an "open" conformation, while the incoming dCTP is still able to bind for the extension reaction. The structure of the mismatched DNA-Polζ ternary complex reveals insights into mechanisms that either stall or favor continued DNA synthesis in eukaryotes.

DOI: https://doi.org/10.1038/s41467-022-28644-7
Nucleic Acids Res. 2022 Feb 22. pii: gkac079. [Epub ahead of print]

NBS1-CtIP-mediated DNA end resection suppresses cGAS binding to micronuclei.

Salim Abdisalaam, Shibani Mukherjee, Souparno Bhattacharya, Sharda Kumari, Debapriya Sinha, Janice Ortega, Guo-Min Li, Hesham A Sadek, Sunil Krishnan, Aroumougame Asaithamby.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor-in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor-functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head-associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1's interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.

DOI: https://doi.org/10.1093/nar/gkac079
Cell Death Differ. 2022 Feb 22.

Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.

Joanna Maria Merchut-Maya, Jiri Bartek, Jirina Bartkova, Panagiotis Galanos, Mattia Russel Pantalone, MyungHee Lee, Huanhuan L Cui, Patrick J Shilling, Christian Beltoft Brøchner, Helle Broholm, Apolinar Maya-Mendoza, Cecilia Söderberg-Naucler, Jiri Bartek.

Viral infections enhance cancer risk and threaten host genome integrity. Although human cytomegalovirus (HCMV) proteins have been detected in a wide spectrum of human malignancies and HCMV infections have been implicated in tumorigenesis, the underlying mechanisms remain poorly understood. Here, we employed a range of experimental approaches, including single-molecule DNA fiber analysis, and showed that infection by any of the four commonly used HCMV strains: AD169, Towne, TB40E or VR1814 induced replication stress (RS), as documented by host-cell replication fork asymmetry and formation of 53BP1 foci. The HCMV-evoked RS triggered an ensuing host DNA damage response (DDR) and chromosomal instability in both permissive and non-permissive human cells, the latter being particularly relevant in the context of tumorigenesis, as such cells can survive and proliferate after HCMV infection. The viral major immediate early enhancer and promoter (MIEP) that controls expression of the viral genes IE72 (IE-1) and IE86 (IE-2), contains transcription-factor binding sites shared by promoters of cellular stress-response genes. We found that DNA damaging insults, including those relevant for cancer therapy, enhanced IE72/86 expression. Thus, MIEP has been evolutionary shaped to exploit host DDR. Ectopically expressed IE72 and IE86 also induced RS and increased genomic instability. Of clinical relevance, we show that undergoing standard-of-care genotoxic radio-chemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV protein markers after tumor recurrence. Collectively, these results are consistent with our proposed concept of HCMV hijacking transcription-factor binding sites shared with host stress-response genes. We present a model to explain the potential oncomodulatory effects of HCMV infections through enhanced replication stress, subverted DNA damage response and induced genomic instability.

DOI: https://doi.org/10.1038/s41418-022-00953-w
Nat Commun. 2022 Feb 23. 13(1): 1015

Small Cajal body-associated RNA 2 (scaRNA2) regulates DNA repair pathway choice by inhibiting DNA-PK.

Sofie Bergstrand, Eleanor M O'Brien, Christos Coucoravas, Dominika Hrossova, Dimitra Peirasmaki, Sandro Schmidli, Soniya Dhanjal, Chiara Pederiva, Lee Siggens, Oliver Mortusewicz, Julienne J O'Rourke, Marianne Farnebo.

Evidence that long non-coding RNAs (lncRNAs) participate in DNA repair is accumulating, however, whether they can control DNA repair pathway choice is unknown. Here we show that the small Cajal body-specific RNA 2 (scaRNA2) can promote HR by inhibiting DNA-dependent protein kinase (DNA-PK) and, thereby, NHEJ. By binding to the catalytic subunit of DNA-PK (DNA-PKcs), scaRNA2 weakens its interaction with the Ku70/80 subunits, as well as with the LINP1 lncRNA, thereby preventing catalytic activation of the enzyme. Inhibition of DNA-PK by scaRNA2 stimulates DNA end resection by the MRN/CtIP complex, activation of ATM at DNA lesions and subsequent repair by HR. ScaRNA2 is regulated in turn by WRAP53β, which binds this RNA, sequestering it away from DNA-PKcs and allowing NHEJ to proceed. These findings reveal that RNA-dependent control of DNA-PK catalytic activity is involved in regulating whether the cell utilizes NHEJ or HR.

DOI: https://doi.org/10.1038/s41467-022-28646-5
Mol Cell. 2022 Feb 16. pii: S1097-2765(22)00101-0. [Epub ahead of print]

RIF1 acts in DNA repair through phosphopeptide recognition of 53BP1.

Dheva Setiaputra, Cristina Escribano-Díaz, Julia K Reinert, Pooja Sadana, Dali Zong, Elsa Callen, Chérine Sifri, Jan Seebacher, André Nussenzweig, Nicolas H Thomä, Daniel Durocher.

  The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.

Keywords:  53BP1; BRCA1; DNA double-strand breaks; NHEJ; RIF1; class switch recombination; phosphorylation; poly(ADP) ribose polymerase inhibitors; shieldin

DOI:  https://doi.org/10.1016/j.molcel.2022.01.025
Genes (Basel). 2022 Jan 20. pii: 180. [Epub ahead of print]13(2):

Regulation of Replication Stress in Alternative Lengthening of Telomeres by Fanconi Anaemia Protein.

Duda Li, Kailong Hou, Ke Zhang, Shuting Jia.

  Fanconi anaemia (FA)-related proteins function in interstrand crosslink (ICL) repair pathways and multiple damage repair pathways. Recent studies have found that FA proteins are involved in the regulation of replication stress (RS) in alternative lengthening of telomeres (ALT). Since ALT cells often exhibit high-frequency ATRX mutations and high levels of telomeric secondary structure, high levels of DNA damage and replicative stress exist in ALT cells. Persistent replication stress is required to maintain the activity of ALT mechanistically, while excessive replication stress causes ALT cell death. FA proteins such as FANCD2 and FANCM are involved in the regulation of this balance by resolving or inhibiting the formation of telomere secondary structures to stabilize stalled replication forks and promote break-induced repair (BIR) to maintain the survival of ALT tumour cells. Therefore, we review the role of FA proteins in replication stress in ALT cells, providing a rationale and direction for the targeted treatment of ALT tumours.

Keywords:  BRCA1/2; FAMCM; FANCD2; RAD51; alternative lengthening of telomeres (ALT); replication stress

DOI:  https://doi.org/10.3390/genes13020180
J Vis Exp. 2022 Feb 03.

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells using the DNA Fiber Assay.

Davi J Martins, Stephanie Tirman, Annabel Quinet, Carlos F M Menck.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

DOI: https://doi.org/10.3791/63448
Int J Mol Sci. 2022 Feb 17. pii: 2212. [Epub ahead of print]23(4):

A Low-Activity Polymorphic Variant of Human NEIL2 DNA Glycosylase.

Zarina I Kakhkharova, Dmitry O Zharkov, Inga R Grin.

  Human NEIL2 DNA glycosylase (hNEIL2) is a base excision repair protein that removes oxidative lesions from DNA. A distinctive feature of hNEIL2 is its preference for the lesions in bubbles and other non-canonical DNA structures. Although a number of associations of polymorphisms in the hNEIL2 gene were reported, there is little data on the functionality of the encoded protein variants, as follows: only hNEIL2 R103Q was described as unaffected, and R257L, as less proficient in supporting the repair in a reconstituted system. Here, we report the biochemical characterization of two hNEIL2 variants found as polymorphisms in the general population, R103W and P304T. Arg103 is located in a long disordered segment within the N-terminal domain of hNEIL2, while Pro304 occupies a position in the β-turn of the DNA-binding zinc finger motif. Similar to the wild-type protein, both of the variants could catalyze base excision and nick DNA by β-elimination but demonstrated a lower affinity for DNA. Steady-state kinetics indicates that the P304T variant has its catalytic efficiency (in terms of kcat/KM) reduced ~5-fold compared with the wild-type hNEIL2, whereas the R103W enzyme is much less affected. The P304T variant was also less proficient than the wild-type, or R103W hNEIL2, in the removal of damaged bases from single-stranded and bubble-containing DNA. Overall, hNEIL2 P304T could be worthy of a detailed epidemiological analysis as a possible cancer risk modifier.

Keywords:  DNA damage; DNA glycosylases; DNA repair; NEIL2; single-nucleotide polymorphisms; variants of unknown significance

DOI:  https://doi.org/10.3390/ijms23042212
Genes Cells. 2022 Feb 23.

XRCC1 counteracts PARP poisons, Olaparib and Talazoparib, and a clinical alkylating agent, Temozolomide, by promoting the removal of trapped PARP1 from broken DNA.

Kouji Hirota, Masato Ooka, Naoto Shimizu, Kousei Yamada, Masataka Tsuda, Mahmoud Abdelghany Ibrahim, Shintaro Yamada, Hiroyuki Sasanuma, Mitsuko Masutani, Shunichi Takeda.

  Base excision repair (BER) removes damaged bases by generating single-strand breaks (SSBs), gap-filling by DNA polymerase β (POLβ), and resealing SSBs. A base-damaging agent, MMS is widely used to study BER. BER increases cellular tolerance to MMS, anti-cancer base-damaging drugs, Temozolomide, Carmustine, and Lomustine, and to clinical poly(ADP ribose)polymerase (PARP) poisons, Olaparib and Talazoparib. The poisons stabilize PARP1/SSB complexes, inhibiting access of BER factors to SSBs. PARP1 and XRCC1 collaboratively promote SSB resealing by recruiting POLβ to SSBs, but XRCC1-/- cells are much more sensitive to MMS than PARP1-/- cells. We recently report that the PARP1 loss in XRCC1-/- cells restores their MMS tolerance and conclude that XPCC1 facilitates the release of PARP1 from SSBs by maintaining its autoPARylation. We here show that the PARP1 loss in XRCC1-/- cells also restores their tolerance to the three anti-cancer base-damaging drugs, although they and MMS induce different sets of base damage. We reveal the synthetic lethality of the XRCC1-/- mutation, but not POLβ-/- , with Olaparib and Talazoparib, indicating that XRCC1 is a unique BER factor in suppressing toxic PARP1/SSB complex and can suppress even when PARP1 catalysis is inhibited. In conclusion, XRCC1 suppresses the PARP1/SSB complex via PARP1 catalysis-dependent and independent mechanisms.

Keywords:  Carmustine; Lomustine; Olaparib; PARP poison; PARP1; Talazoparib; Temozolomide; XRCC1

DOI:  https://doi.org/10.1111/gtc.12929
Life (Basel). 2022 Feb 12. pii: 271. [Epub ahead of print]12(2):

The Multiple Roles of Glucose-6-Phosphate Dehydrogenase in Tumorigenesis and Cancer Chemoresistance.

Jiaqi Song, Huanran Sun, Shuai Zhang, Changliang Shan.

  The pentose phosphate pathway (PPP) is a branch from glycolysis that begins from glucose-6-phosphate (G6P) and ends up with fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (GADP). Its primary physiological significance is to provide nicotinamide adenine dinucleotide phosphate (NADPH) and nucleotides for vital activities such as reactive oxygen species (ROS) defense and DNA synthesis. Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping protein with 514 amino acids that is also the rate-limiting enzyme of PPP, catalyzing G6P into 6-phosphogluconolactone (6PGL) and producing the first NADPH of this pathway. Increasing evidence indicates that G6PD is upregulated in diverse cancers, and this dysfunction influences DNA synthesis, DNA repair, cell cycle regulation and redox homeostasis, which provides advantageous conditions for cancer cell growth, epithelial-mesenchymal transition (EMT), invasion, metastasis and chemoresistance. Thus, targeting G6PD by inhibitors has been shown as a promising strategy in treating cancer and reversing chemotherapeutic resistance. In this review, we will summarize the existing knowledge concerning G6PD and discuss its role, regulation and inhibitors in cancer development and chemotherapy resistance.

Keywords:  G6PD; chemotherapy resistance; enzyme; inhibitor; metabolism; tumorigenesis

DOI:  https://doi.org/10.3390/life12020271
Genes (Basel). 2022 Jan 18. pii: 166. [Epub ahead of print]13(2):

Stalling of Eukaryotic Translesion DNA Polymerases at DNA-Protein Cross-Links.

Anna V Yudkina, Evgeniy S Shilkin, Alena V Makarova, Dmitry O Zharkov.

  DNA-protein cross-links (DPCs) are extremely bulky adducts that interfere with replication. In human cells, they are processed by SPRTN, a protease activated by DNA polymerases stuck at DPCs. We have recently proposed the mechanism of the interaction of DNA polymerases with DPCs, involving a clash of protein surfaces followed by the distortion of the cross-linked protein. Here, we used a model DPC, located in the single-stranded template, the template strand of double-stranded DNA, or the displaced strand, to study the eukaryotic translesion DNA polymerases ζ (POLζ), ι (POLι) and η (POLη). POLι demonstrated poor synthesis on the DPC-containing substrates. POLζ and POLη paused at sites dictated by the footprints of the polymerase and the cross-linked protein. Beyond that, POLζ was able to elongate the primer to the cross-link site when a DPC was in the template. Surprisingly, POLη was not only able to reach the cross-link site but also incorporated 1-2 nucleotides past it, which makes POLη the most efficient DNA polymerase on DPC-containing substrates. However, a DPC in the displaced strand was an insurmountable obstacle for all polymerases, which stalled several nucleotides before the cross-link site. Overall, the behavior of translesion polymerases agrees with the model of protein clash and distortion described above.

Keywords:  DNA polymerases; DNA replication; DNA–protein cross-link; translesion synthesis

DOI:  https://doi.org/10.3390/genes13020166
Cancers (Basel). 2022 Feb 14. pii: 953. [Epub ahead of print]14(4):

Everything Comes with a Price: The Toxicity Profile of DNA-Damage Response Targeting Agents.

Federica Martorana, Leandro Apolinario Da Silva, Cristiana Sessa, Ilaria Colombo.

  Targeting the inherent vulnerability of cancer cells with an impaired DNA Damage Repair (DDR) machinery, Poly-ADP-Ribose-Polymerase (PARP) inhibitors have yielded significant results in several tumor types, eventually entering clinical practice for the treatment of ovarian, breast, pancreatic and prostate cancer. More recently, inhibitors of other key components of DNA repair, such as ATR, CHK1 and WEE1, have been developed and are currently under investigation in clinical trials. The inhibition of DDR inevitably induces on-target and off-target adverse events. Hematological and gastrointestinal toxicities as well as fatigue are common with all DDR-targeting agents, while other adverse events are drug specific, such as hypertension with niraparib and transaminase elevation with rucaparib. Cases of pneumonitis and secondary hematological malignancies have been reported with PARP inhibitors and, despite being overly rare, they deserve particular attention due to their severity. Safety also represents a crucial issue for the development of combination regimens incorporating DDR-targeting agents with other treatments, such as chemotherapy, anti-angiogenics or immunotherapy. As such, overlapping and cumulative toxicities should be considered, especially when more than two classes of drugs are combined. Here, we review the safety profile of DDR-targeting agents when used as single agents or in combination and we provide principles of toxicity management.

Keywords:  ATR inhibitors; CHK1 inhibitors; DNA damage response inhibitors; PARP inhibitors; WEE1 inhibitors; adverse events; safety profile

DOI:  https://doi.org/10.3390/cancers14040953
Nat Commun. 2022 Feb 25. 13(1): 1059

A mechanism of origin licensing control through autoinhibition of S. cerevisiae ORC·DNA·Cdc6.

Jan Marten Schmidt, Ran Yang, Ashish Kumar, Olivia Hunker, Jan Seebacher, Franziska Bleichert.

The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to DNA initiates the ATP-dependent recruitment of Cdc6, Cdt1 and Mcm2-7 loading, but the structural details for timely ATPase site regulation and for how loading can be impeded by inhibitory signals, such as cyclin-dependent kinase phosphorylation, are unknown. Using cryo-electron microscopy, we have determined several structures of S. cerevisiae ORC·DNA·Cdc6 intermediates at 2.5-2.7 Å resolution. These structures reveal distinct ring conformations of the initiator·co-loader assembly and inactive ATPase site configurations for ORC and Cdc6. The Orc6 N-terminal domain laterally engages the ORC·Cdc6 ring in a manner that is incompatible with productive Mcm2-7 docking, while deletion of this Orc6 region alleviates the CDK-mediated inhibition of Mcm7 recruitment. Our findings support a model in which Orc6 promotes the assembly of an autoinhibited ORC·DNA·Cdc6 intermediate to block origin licensing in response to CDK phosphorylation and to avert DNA re-replication.

DOI: https://doi.org/10.1038/s41467-022-28695-w
Bone Res. 2022 Feb 24. 10(1): 19

RIOX1-demethylated cGAS regulates ionizing radiation-elicited DNA repair.

Yanxuan Xiao, Jingyi Li, Xiaoyu Liao, Yumin He, Tao He, Cuiping Yang, Lu Jiang, So Mi Jeon, Jong-Ho Lee, Yongbin Chen, Rui Liu, Qianming Chen.

Exposure to radiation causes DNA damage; hence, continuous surveillance and timely DNA repair are important for genome stability. Epigenetic modifications alter the chromatin architecture, thereby affecting the efficiency of DNA repair. However, how epigenetic modifiers coordinate with the DNA repair machinery to modulate cellular radiosensitivity is relatively unknown. Here, we report that loss of the demethylase ribosomal oxygenase 1 (RIOX1) restores cell proliferation and reduces cell death after exposure to ionizing radiation. Furthermore, RIOX1 depletion enhances homologous recombination (HR) repair but not nonhomologous end-joining (NHEJ) repair in irradiated bone marrow cells and oral mucosal epithelial cells. Mechanistic study demonstrates that RIOX1 removes monomethylation at K491 of cyclic GMP-AMP synthase (cGAS) to release cGAS from its interaction with the methyl-lysine reader protein SAGA complex-associated factor 29 (SGF29), which subsequently enables cGAS to interact with poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase 1 (PARP1) at DNA break sites, thereby blocking PARP1-mediated recruitment of Timeless. High expression of RIOX1 maintains cGAS K491me at a low level, which impedes HR repair and reduces cellular tolerance to ionizing radiation. This study highlights a novel RIOX1-dependent mechanism involved in the non-immune function of cGAS that is essential for the regulation of ionizing radiation-elicited HR repair.

DOI: https://doi.org/10.1038/s41413-022-00194-0
Cancers (Basel). 2022 Feb 11. pii: 897. [Epub ahead of print]14(4):

Precision Medicine for BRCA/PALB2-Mutated Pancreatic Cancer and Emerging Strategies to Improve Therapeutic Responses to PARP Inhibition.

Daniel R Principe.

  Pancreatic cancer is projected to become the second leading cause of cancer-related death by 2030. As patients typically present with advanced disease and show poor responses to broad-spectrum chemotherapy, overall survival remains a dismal 10%. This underscores an urgent clinical need to identify new therapeutic approaches for PDAC patients. Precision medicine is now the standard of care for several difficult-to-treat cancer histologies. Such approaches involve the identification of a clinically actionable molecular feature, which is matched to an appropriate targeted therapy. Selective poly (ADP-ribose) polymerase (PARP) inhibitors such as Niraparib, Olaparib, Talazoparib, Rucaparib, and Veliparib are now approved for several cancers with loss of high-fidelity double-strand break homologous recombination (HR), namely those with deleterious mutations to BRCA1/2, PALB2, and other functionally related genes. Recent evidence suggests that the presence of such mutations in pancreatic ductal adenocarcinoma (PDAC), the most common and lethal pancreatic cancer histotype, significantly alters drug responses both with respect to first-line chemotherapy and maintenance therapy. In this review, we discuss the current treatment paradigm for PDAC tumors with confirmed deficits in double-strand break HR, as well as emerging strategies to both improve responses to PARP inhibition in HR-deficient PDAC and confer sensitivity to tumors proficient in HR repair.

Keywords:  BRCA; PALB2; PARP inhibitor; homologous recombination deficiency; pancreatic ductal adenocarcinoma; precision medicine

DOI:  https://doi.org/10.3390/cancers14040897
Semin Cell Dev Biol. 2022 Feb 21. pii: S1084-9521(22)00052-0. [Epub ahead of print]

Ubiquitin and SUMO as timers during DNA replication.

Rodrigo Martín-Rufo, Guillermo de la Vega-Barranco, Emilio Lecona.

  Every time a cell copies its DNA the genetic material is exposed to the acquisition of mutations and genomic alterations that corrupt the information passed on to daughter cells. A tight temporal regulation of DNA replication is necessary to ensure the full copy of the DNA while preventing the appearance of genomic instability. Protein modification by ubiquitin and SUMO constitutes a very complex and versatile system that allows the coordinated control of protein stability, activity and interactome. In chromatin, their action is complemented by the AAA+ ATPase VCP/p97 that recognizes and removes ubiquitylated and SUMOylated factors from specific cellular compartments. The concerted action of the ubiquitin/SUMO system and VCP/p97 determines every step of DNA replication enforcing the ordered activation/inactivation, loading/unloading and stabilization/destabilization of replication factors. Here we analyze the mechanisms used by ubiquitin/SUMO and VCP/p97 to establish molecular timers throughout DNA replication and their relevance in maintaining genome stability. We propose that these PTMs are the main molecular watch of DNA replication from origin recognition to replisome disassembly.

Keywords:  DNA damage; DNA replication; Genomic instability; Molecular timer; SUMO; Ubiquitin; VCP/p97

DOI:  https://doi.org/10.1016/j.semcdb.2022.02.013
Oncogene. 2022 Feb 21.

p53-NEIL1 co-abnormalities induce genomic instability and promote synthetic lethality with Chk1 inhibition in multiple myeloma having concomitant 17p13(del) and 1q21(gain).

Phaik Ju Teoh, Omer An, Tae-Hoon Chung, Thamil Vaiyapuri, Anandhkumar Raju, Michal M Hoppe, Sabrina H M Toh, Wilson Wang, Ming Chun Chan, Melissa J Fullwood, Anand D Jeyasekharan, Vinay Tergaonkar, Leilei Chen, Henry Yang, Wee Joo Chng.

Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.

DOI: https://doi.org/10.1038/s41388-022-02227-8
Bioorg Med Chem. 2022 Mar 15. pii: S0968-0896(21)00585-X. [Epub ahead of print]58 116577

Examination of multiple Trypanosoma cruzi targets in a new drug discovery approach for Chagas disease.

Iván Beltran-Hortelano, Verónica Alcolea, María Font, Silvia Pérez-Silanes.

  Chagas disease (CD) is a centenarian neglected parasitosis caused by the protozoan Trypanosoma cruzi (T. cruzi). Despite the continuous efforts of many organizations and institutions, CD is still an important human health problem worldwide. A lack of a safe and affordable treatment has led drug discovery programmes to focus, for years, on the search for molecules enabling interference with enzymes that are essential for T. cruzi survival. In this work, the authors want to offer a brief overview of the different validated targets that are involved in diverse parasite pathways: glycolysis, sterol synthesis, the de novo biosynthesis of pyrimidine nucleotides, the degradative processing of peptides and proteins, oxidative stress damage and purine salvage and nucleotide synthesis and metabolism. Their structural aspects, function, active sites, etc. were studied and considered with the aim of defining molecular bases in the search for new effective treatments for CD. This review also compiles, as much as possible, all the inhibitors reported to date against these T. cruzi targets, serving as a reference for future research in this field.

Keywords:  CYP51; Cruzain; Dihydrofolate reductase; Dihydroorotate dehydrogenase; Pteridine reductase; Sterol 14α-demethylase; Superoxide dismutase; Thymidylate synthase; Triosephosphate isomerase; Trypanosoma cruzi; Trypanothione reductase

DOI:  https://doi.org/10.1016/j.bmc.2021.116577
Biochem Soc Trans. 2022 Feb 22. pii: BST20210446. [Epub ahead of print]

IMPDH dysregulation in disease: a mini review.

Anika L Burrell, Justin M Kollman.

  Inosine-5'-monophosphate dehydrogenase (IMPDH) is a highly conserved enzyme in purine metabolism that is tightly regulated on multiple levels. IMPDH has a critical role in purine biosynthesis, where it regulates flux at the branch point between adenine and guanine nucleotide synthesis, but it also has a role in transcription regulation and other moonlighting functions have been described. Vertebrates have two isoforms, IMPDH1 and IMPDH2, and point mutations in each are linked to human disease. Mutations in IMPDH2 in humans are associated with neurodevelopmental disease, but the effects of mutations at the enzyme level have not yet been characterized. Mutations in IMPDH1 lead to retinal degeneration in humans, and recent studies have characterized how they cause functional defects in regulation. IMPDH1 is expressed as two unique splice variants in the retina, a tissue with very high and specific demands for purine nucleotides. Recent studies have revealed functional differences among splice variants, demonstrating that retinal variants up-regulate guanine nucleotide synthesis by reducing sensitivity to feedback inhibition by downstream products. A better understanding of the role of IMPDH1 in the retina and the characterization of an animal disease model will be critical for determining the molecular mechanism of IMPDH1-associated blindness.

Keywords:  allosteric regulation; retinitis pigmentosa; splice variants

DOI:  https://doi.org/10.1042/BST20210446
Nucleic Acids Res. 2022 Feb 25. pii: gkac118. [Epub ahead of print]

Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR.

Zhihan Zhao, Peng Shang, Fanny Sage, Niels Geijsen.

CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy 'Ligation-Assisted Homologous Recombination' (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.

DOI: https://doi.org/10.1093/nar/gkac118
Nucleosides Nucleotides Nucleic Acids. 2022 Feb 20. 1-22

Protein-lipid interactions of human dihydroorotate dehydrogenase and three mutants associated with Miller syndrome.

Juan Manuel Orozco Rodriguez, Hanna Wacklin-Knecht, Wolfgang Knecht.

  Human dihydroorotate dehydrogenase (DHODH) catalyzes the fourth step of the de novo pyrimidine biosynthesis pathway and uses ubiquinone Q10, a lipophilic molecule located in the inner mitochondrial membrane (IMM), as its co-substrate. DHODH is anchored to the IMM by a single transmembrane helix located at its N-terminus. Nevertheless, how DHODH function is determined by its surrounding membrane environment and protein-lipid interactions, as well as the mechanism by which ubiquinone Q10 accesses the active site of DHODH from within the membrane are still largely unknown. Here, we describe the interaction between wild-type DHODH and three DHODH mutants associated with Miller syndrome and lipids using enzymatic assays, thermal stability assays and Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). Our results provide evidence indicating that the N-terminal part of human DHODH is not only a structural element for mitochondrial import and location of DHODH, but also influences enzymatic activity and utilization of ubiquinone Q10 and ubiquinone analogues in in vitro assays. They also support the role of tetraoleoyl cardiolipin as a lipid interacting with DHODH. Additionally, the results from QCM-D show that the Miller syndrome mutants studied differ in their interactions with supported lipid bilayers compared to wild-type DHODH. These altered interactions add another dimension to the effects of mutations found in Miller syndrome. To the best of our knowledge, this is the first investigation of the protein-lipid interactions of DHODH variants associated with Miller syndrome.

Keywords:  Mendelian disorders; Pyrimidine biosynthesis; QCM-D; protein-lipid interactions; ubiquinone

DOI:  https://doi.org/10.1080/15257770.2022.2039393
Mol Genet Metab. 2022 Feb 19. pii: S1096-7192(22)00139-1. [Epub ahead of print]

Inborn errors of purine and pyrimidine metabolism: A guide to diagnosis.

Agnieszka Jurecka, Anna Tylki-Szymanska.

  Inborn errors of purine and pyrimidine (P/P) metabolism are under-reported and rarely mentioned in the general literature or in clinical practice, as well as in reviews dedicated to other inborn errors of metabolism (IEMs). However, their diagnosis is important because genetic counseling can be provided and, in some cases, specific treatment exists that may slow or even reverse clinical signs. The purpose of this review is to provide a practical guideline on the suspicion and investigation of inborn errors of P/P metabolism. Failure of a physician to recognize the presence of these disorders may be devastating for affected infants and children because of its permanent effects in the patient, and for their parents because of implications for future offspring. Diagnosis is crucial because genetic counseling can be provided and, in some cases, specific treatment can be offered that may slow or even reverse clinical symptoms. This review highlights the risk factors in the history, the important examination findings, and the appropriate biochemical investigation of the child. Herein we describe the approach to the diagnosis of P/P disorders and emphasize clinical situations in which physicians should consider these diseases as diagnostic possibilities.

Keywords:  Encephalopathy; Hypotonia; Myopathy; Purines; Pyrimidines; Seizures

DOI:  https://doi.org/10.1016/j.ymgme.2022.02.007