RTEL1 helicase counteracts RAD51-mediated homologous recombination and fork reversal to safeguard replicating genomes.

Homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSBs), replication stress responses, and genome maintenance. However, unregulated HR during replication can impair genome duplication and compromise genome stability. The mechanisms underlying HR regulation during DNA replication are obscure.

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication.

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments.

Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint.

The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase-polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling.

Homologous Recombination as a Fundamental Genome Surveillance Mechanism during DNA Replication.

Accurate and complete genome replication is a fundamental cellular process for the proper transfer of genetic material to cell progenies, normal cell growth, and genome stability. However, a plethora of extrinsic and intrinsic factors challenge individual DNA replication forks and cause replication stress (RS), a hallmark of cancer. When challenged by RS, cells deploy an extensive range of mechanisms to safeguard replicating genomes and limit the burden of DNA damage.

Homology-directed repair protects the replicating genome from metabolic assaults.

Homology-directed repair (HDR) safeguards DNA integrity under various forms of stress, but how HDR protects replicating genomes under extensive metabolic alterations remains unclear. Here, we report that besides stalling replication forks, inhibition of ribonucleotide reductase (RNR) triggers metabolic imbalance manifested by the accumulation of increased reactive oxygen species (ROS) in cell nuclei. This leads to a redox-sensitive activation of the ATM kinase followed by phosphorylation of the MRE11 nuclease, which in HDR-deficient settings degrades stalled replication forks.

Equilibrium between nascent and parental MCM proteins protects replicating genomes.

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes.

Physiological Tolerance to ssDNA Enables Strand Uncoupling during DNA Replication.

It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress.

ATR Signaling Uncouples the Role of RAD51 Paralogs in Homologous Recombination and Replication Stress Response.

ATR kinase-mediated replication checkpoint is vital for genome maintenance following replication stress. Previously, we showed that XRCC2-RAD51D (DX2) sub-complex of RAD51 paralogs restrains active DNA synthesis during dNTP alterations, in a manner dependent on ATR-mediated phosphorylation of XRCC2. Here, we find that unrestrained fork progression in XRCC2 deficiency and phosphorylation defect causes replication-associated errors, subsequently resulting in genome-wide double-strand breaks (DSBs) and early activation of ATM signaling.

53BP1 nuclear bodies enforce replication timing at under-replicated DNA to limit heritable DNA damage.

Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown.

XRCC2 Regulates Replication Fork Progression during dNTP Alterations.

RAD51 paralogs are essential for maintenance of genomic integrity through protection of stalled replication forks and homology-directed repair (HDR) of double-strand breaks. Here, we find that a subset of RAD51 paralogs, XRCC2 (FANCU) and its binding partner RAD51D, restrain active DNA synthesis during dinucleotide triphosphate (dNTP) alterations in a manner independent of HDR. The absence of XRCC2 is associated with increased levels of RRM2, the regulatory subunit of ribonucleotide reductase (RNR), and concomitantly high nucleotide pools, leading to unrestrained fork progression and accumulation of DNA damage during dNTP alterations.

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process.

Redox-sensitive alteration of replisome architecture safeguards genome integrity.

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS.

FANCJ helicase controls the balance between short- and long-tract gene conversions between sister chromatids.

The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease.

53BP1 fosters fidelity of homology-directed DNA repair.

Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52.

Trans-dichlorooxovandium (IV) complex as a novel photoinducible DNA interstrand crosslinker for cancer therapy.

Although DNA interstrand crosslinking (ICL) agents such as mitomycin C, cisplatin and psoralen serve as potent anticancer drugs, these agents are known to have dose-limiting toxic effects on normal cells. Moreover, tumor resistance to these agents has been reported. Here, we show that trans-dichlorooxovanadium (IV) complex of pyrenyl terpyridine (VDC) is a novel photoinducible DNA crosslinking agent.

Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart.

Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs.

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide.

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected.

Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase.

Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function.

Oxovanadium(IV) complexes of curcumin for cellular imaging and mitochondria targeted photocytotoxicity.

Oxovanadium(IV) complexes [VO(R-tpy)(cur)](ClO4) (1, 2) of curcumin (Hcur) and terpyridine ligands (R-tpy) where R is phenyl (phtpy in 1) or p-triphenylphosphonium methylphenyl bromide (C6H4CH2PPh3Br) (TPP-phtpy in 2) were prepared and characterized and their DNA photocleavage activity, photocytotoxicity and cellular localization in cancer cells (HeLa and MCF-7) were studied. Acetylacetonate (acac) complexes [VO(R-tpy)(acac)](ClO4) of phtpy (3) and TPP-phtpy (4) were prepared and used as the control species. These complexes showed efficient cleavage of pUC19 DNA in visible light of 454 nm and near-IR light of 705 nm.

Carbohydrate-appended photocytotoxic (imidazophenanthroline)-oxovanadium(IV) complexes for cellular targeting and imaging.

Oxovanadium(IV) complexes [VO(aip)(L)](ClO4)2 (L = phtpy, 1; stpy, 2) and [VO(pyip)(L)](ClO4)2 (L = phtpy, 3; stpy, 4), where aip is 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline, pyip is [2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, phtpy is (4'-phenyl)-2,2':6',2''-terpyridine and stpy is (2,2':6',2''-terpyridin-4'-oxy)ethyl-β-D-glucopyranoside, were prepared, characterized and their DNA binding and photocleavage activity, cellular uptake and photocytotoxicity in visible light were studied. The complexes are avid binders to calf thymus DNA (K(b) ~10(5) mol(-1)). They efficiently cleave pUC19 DNA in red light of 705 nm via the formation of HO˙ species.

ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair.

The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway.

Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M.