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DNA damage tolerance pathways that allow for the completion of replication following fork arrest are critical in maintaining genome stability during cell division. The main DNA damage tolerance pathways include strand switching, replication fork reversal and translesion synthesis (TLS). The TLS pathway is mediated by specialised DNA polymerases that can accommodate altered DNA structures during DNA synthesis, and are important in allowing replication to proceed after fork arrest, preventing fork collapse that can generate more deleterious double-strand breaks in the genome. TLS may occur directly at the fork, or at gaps remaining behind the fork, in the process of post-replication repair. Inactivating mutations in the human POLH gene encoding the Y-family DNA polymerase Pol η causes the skin cancer-prone genetic disease xeroderma pigmentosum variant (XPV). Pol η also contributes to chemoresistance during cancer treatment by bypassing DNA lesions induced by anti-cancer drugs including cisplatin. We review the current understanding of the canonical role of Pol η in translesion synthesis following replication arrest, as well as a number of emerging non-canonical roles of the protein in other aspects of DNA metabolism. [ABSTRACT FROM AUTHOR]

Poultry immunoglobulin genes undergo diversification through homologous recombination (HR) and somatic hypermutation (SHM). Most animals share a similar system in immunoglobulin diversification, with the rare exception that human and murine immunoglobulin genes diversify through V(D)J recombination. Poultry possesses only one functional variable gene for each immunoglobulin heavy (HC) and light chains (LC), with clusters of nonproductive pseudogenes upstream. During the B cell development, the functional variable gene is overwritten by sequences from the pseudo-variable genes via a process known as gene conversion (GC), a kind of HR. Point mutations caused in the functional variable gene also contribute to immunoglobulin diversification. This review discusses the latest findings on the molecular mechanisms of antibody gene diversification in poultry, using chickens as a model. Additionally, it will outline how these basic research findings have recently been applied especially in the medical field. [ABSTRACT FROM AUTHOR]

The fidelity of replication, especially in the presence of DNA damage, is essential for the proper function of cells. Mutations that inactivate genes involved in DNA damage repair or bypass are enriched in several types of cancer cells. Thus, it is important to further our understanding of the mechanisms governing replication fidelity. PCNA is a ring-shaped complex that encircles DNA at the front of the replication fork, at the double-stranded/single-stranded DNA junction. It serves as a processivity factor for the different DNA replication polymerases, allowing them to replicate longer stretches of DNA by physically tethering them to the DNA and preventing their detachment. In addition, PCNA also regulates and coordinates different DNA damage bypass pathways meant to allow DNA replication in the presence of DNA damage. Due to its essentiality and the numerous functions it has in the cell, much is still unclear about PCNA. Here, we utilize PCNA mutants that lower the stability of the PCNA complex on the chromatin, and thus tend to disassociate and fall from the DNA. Using these mutants, we show that PCNA's physical presence on the DNA can prevent DNA misalignment at repetitive sequences, leading to increased mutation formation. We also show that PCNA-interacting proteins play an important role in strengthening the ring's stability on the chromatin. Such repetitive sequence-induced mutations are common in several human diseases and it is important to study their formation and the mechanisms guarding against them. [ABSTRACT FROM AUTHOR]

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein–protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies. [ABSTRACT FROM AUTHOR]

Accurate DNA replication and transcription elongation are crucial for preventing the accumulation of unreplicated DNA and genomic instability. Cells have evolved multiple mechanisms to deal with impaired replication fork progression, challenged by both intrinsic and extrinsic impediments. The bacterium Bacillus subtilis , which adopts multiple forms of differentiation and development, serves as an excellent model system for studying the pathways required to cope with replication stress to preserve genomic stability. This review focuses on the genetics, single molecule choreography, and biochemical properties of the proteins that act to circumvent the replicative arrest allowing the resumption of DNA synthesis. The RecA recombinase, its mediators (RecO, RecR, and RadA/Sms) and modulators (RecF, RecX, RarA, RecU, RecD2, and PcrA), repair licensing (DisA), fork remodelers (RuvAB, RecG, RecD2, RadA/Sms, and PriA), Holliday junction resolvase (RecU), nucleases (RnhC and DinG), and translesion synthesis DNA polymerases (PolY1 and PolY2) are key functions required to overcome a replication stress, provided that the fork does not collapse. [ABSTRACT FROM AUTHOR]

Replication fork arrest-induced DNA double strand breaks (DSBs) caused by lesions are effectively suppressed in cells due to the presence of a specialized mechanism, commonly referred to as DNA damage tolerance (DDT). In eukaryotic cells, DDT is facilitated through translesion DNA synthesis (TLS) carried out by a set of DNA polymerases known as TLS polymerases. Another parallel mechanism, referred to as homology-directed DDT, is error-free and involves either template switching or fork reversal. The significance of the DDT pathway is well established. Several diseases have been attributed to defects in the TLS pathway, caused either by mutations in the TLS polymerase genes or dysregulation. In the event of a replication fork encountering a DNA lesion, cells switch from high-fidelity replicative polymerases to low-fidelity TLS polymerases, which are associated with genomic instability linked with several human diseases including, cancer. The role of TLS polymerases in chemoresistance has been recognized in recent years. In addition to their roles in the DDT pathway, understanding noncanonical functions of TLS polymerases is also a key to unraveling their importance in maintaining genomic stability. Here we summarize the current understanding of TLS pathway in DDT and its implication for human health. [ABSTRACT FROM AUTHOR]

DNA replication is a tightly controlled process that ensures the faithful duplication of the genome. However, DNA damage arising from both endogenous and exogenous assaults gives rise to DNA replication stress associated with replication fork slowing or stalling. Therefore, protecting the stressed fork while prompting its recovery to complete DNA replication is critical for safeguarding genomic integrity and cell survival. Specifically, the plasticity of the replication fork in engaging distinct DNA damage tolerance mechanisms, including fork reversal, repriming, and translesion DNA synthesis, enables cells to overcome a variety of replication obstacles. Furthermore, stretches of single-stranded DNA generated upon fork stalling trigger the activation of the ATR kinase, which coordinates the cellular responses to replication stress by stabilizing the replication fork, promoting DNA repair, and controlling cell cycle and replication origin firing. Deregulation of the ATR checkpoint and aberrant levels of chronic replication stress is a common characteristic of cancer and a point of vulnerability being exploited in cancer therapy. Here, we discuss the various adaptive responses of a replication fork to replication stress and the roles of ATR signaling that bring fork stabilization mechanisms together. We also review how this knowledge is being harnessed for the development of checkpoint inhibitors to trigger the replication catastrophe of cancer cells. [ABSTRACT FROM AUTHOR]

Rev7 is a versatile HORMA (Hop1, Rev7, Mad2) family adaptor protein with multiple roles in mitotic regulation and DNA damage response, and an essential accessory subunit of the translesion synthesis (TLS) DNA polymerase Polζ employed in replication of damaged DNA. Within Polζ, the two copies of Rev7 interact with the two Rev7-bonding motifs (RBM1 and RBM2) of the catalytic subunit Rev3 by a mechanism characteristic of HORMA proteins whereby the "safety-belt" loop of Rev7 closes on the top of the ligand. Here we report the nearly complete backbone and Ile, Val, Leu side-chain methyl NMR resonance assignments of the 27 kDa human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes (BMRB deposition numbers 51651 and 51652) that will facilitate future NMR studies of Rev7 dynamics and interactions. [ABSTRACT FROM AUTHOR]

Several DNA polymerases participate in DNA synthesis during genome replication and DNA repair. PCNA, a homotrimeric ring, acts as a processivity factor for DNA polymerases. PCNA also acts as a "landing pad" for proteins that interact with chromatin and DNA at the moving fork. The interaction between PCNA and polymerase delta (Polδ) is mediated by PIPs (PCNA-interacting peptides), in particular the one on Pol32, a regulatory subunit of Polδ. Here, we demonstrate that pol3-01, an exonuclease mutant of Polδ's catalytic subunit, exhibits a weak interaction with Pol30 compared to the WT DNA polymerase. The weak interaction activates DNA bypass pathways, leading to increased mutagenesis and sister chromatid recombination. Strengthening pol3-01′s weak interaction with PCNA suppresses most of the phenotypes. Our results are consistent with a model in which Pol3-01 tends to detach from the chromatin, allowing an easier replacement of Polδ by the trans-lesion synthesis polymerase Zeta (Polz), thus leading to the increased mutagenic phenotype. [ABSTRACT FROM AUTHOR]

Timely and accurate DNA replication is critical for safeguarding genome integrity and ensuring cell viability. Yet, this process is challenged by DNA damage blocking the progression of the replication machinery. To counteract replication fork stalling, evolutionary conserved DNA damage tolerance (DDT) mechanisms promote DNA damage bypass and fork movement. One of these mechanisms involves "skipping" DNA damage through repriming downstream of the lesion, leaving single-stranded DNA (ssDNA) gaps behind the advancing forks (also known as post-replicative gaps). In vertebrates, repriming in damaged leading templates is proposed to be mainly promoted by the primase and polymerase PRIMPOL. In this review, we discuss recent advances towards our understanding of the physiological and pathological conditions leading to repriming activation in human models, revealing a regulatory network of PRIMPOL activity. Upon repriming by PRIMPOL, post-replicative gaps formed can be filled-in by the DDT mechanisms translesion synthesis and template switching. We discuss novel findings on how these mechanisms are regulated and coordinated in time to promote gap filling. Finally, we discuss how defective gap filling and aberrant gap expansion by nucleases underlie the cytotoxicity associated with post-replicative gap accumulation. Our increasing knowledge of this repriming mechanism – from gap formation to gap filling – is revealing that targeting the last step of this pathway is a promising approach to exploit post-replicative gaps in anti-cancer therapeutic strategies. • Repriming ensures replication of damaged templates, but generates ssDNA gaps. • ssDNA gaps are induced by different sources, both physiological and pathological. • ssDNA gaps are filled in by DNA damage tolerance mechanisms. • Defects in gap filling and gap protection from nucleases may lead to gap toxicity. • Gap filling is a promising target for cancer therapy. [ABSTRACT FROM AUTHOR]

At the core of cellular life lies a carefully orchestrated interplay of DNA replication, recombination, chromatin assembly, sister-chromatid cohesion and transcription. These fundamental processes, while seemingly discrete, are inextricably linked during genome replication. A set of replisome factors integrate various DNA transactions and contribute to the transient formation of sister chromatid junctions involving either the cohesin complex or DNA four-way junctions. The latter structures serve DNA damage bypass and may have additional roles in replication fork stabilization or in marking regions of replication fork blockage. Here, we will discuss these concepts based on the ability of one replisome component, Ctf4, to act as a hub and functionally link these processes during DNA replication to ensure genome maintenance. • Ctf4 is a replisome hub connecting DNA replication with other metabolism processes. • Ctf4 interacting peptides (CIP) and other domains link replication factors to Ctf4. • Ctf4 interactions contribute to cohesion, recombination and chromatin structure. [ABSTRACT FROM AUTHOR]

Cells are constantly exposed to endogenous and exogenous chemical and physical agents that damage their genome by forming DNA lesions. These lesions interfere with the normal functions of DNA such as transcription and replication, and need to be either repaired or tolerated. DNA lesions are accurately removed via various repair pathways. In contrast, tolerance mechanisms do not remove lesions but only allow replication to proceed despite the presence of unrepaired lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS), which is an error-prone strategy and an accurate strategy based on homologous recombination (homology-dependent gap repair [HDGR]). Thus, the mutation frequency reflects the relative extent to which the two tolerance pathways operate in vivo. In the present paper, we review the present understanding of the mechanisms of TLS and HDGR and propose a novel and comprehensive view of the way both strategies interact and are regulated in vivo.

RAD54L is a DNA motor protein with multiple roles in homologous recombination DNA repair. In vitro, RAD54L was shown to also catalyze the reversal and restoration of model replication forks. In cells, however, little is known about how RAD54L may regulate the dynamics of DNA replication. Here, we show that RAD54L restrains the progression of replication forks and functions as a fork remodeler in human cancer cell lines and non-transformed cells. Analogous to HLTF, SMARCAL1 and FBH1, and consistent with a role in fork reversal, RAD54L decelerates fork progression in response to replication stress and suppresses the formation of replication-associated ssDNA gaps. Interestingly, loss of RAD54L prevents nascent strand DNA degradation in both BRCA1/2- and 53BP1-deficient cells, suggesting that RAD54L functions in both pathways of RAD51-mediated replication fork reversal. In the HLTF/SMARCAL1 pathway, RAD54L is critical, but its ability to catalyze branch migration is dispensable, indicative of its function downstream of HLTF/SMARCAL1. Conversely, in the FBH1 pathway, branch migration activity of RAD54L is essential, and FBH1 engagement is dependent on its concerted action with RAD54L. Collectively, our results reveal disparate requirements for RAD54L in two distinct RAD51-mediated fork reversal pathways, positing its potential as a future therapeutic target., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη) were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

The sliding clamp PCNA is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway. Focusing on Saccharomyces cerevisiae budding yeast, we summarized PCNA related-DDT and repair mechanisms that coordinately sustain genome stability and cell survival. In addition, we compared PCNA sequences from various fungal pathogens, considering recent advances in structural features. Importantly, the identification of PCNA epitopes may lead to potential fungal targets for antifungal drug development. [ABSTRACT FROM AUTHOR]

Poultry immunoglobulin genes undergo diversification through homologous recombination (HR) and somatic hypermutation (SHM). Most animals share a similar system in immunoglobulin diversification, with the rare exception that human and murine immunoglobulin genes diversify through V(D)J recombination. Poultry possesses only one functional variable gene for each immunoglobulin heavy (HC) and light chains (LC), with clusters of non-productive pseudogenes upstream. During the B cell development, the functional variable gene is overwritten by sequences from the pseudo-variable genes via a process known as gene conversion (GC), a kind of HR. Point mutations caused in the functional variable gene also contribute to immunoglobulin diversification. This review discusses the latest findings on the molecular mechanisms of antibody gene diversification in poultry, using chickens as a model. Additionally, it will outline how these basic research findings have recently been applied especially in the medical field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Seo, Hirota and Ohta.)

DNA replication is remarkably accurate with estimates of only a handful of mutations per human genome per cell division cycle. Replication stress caused by DNA lesions, transcription-replication conflicts, and other obstacles to the replication machinery must be efficiently overcome in ways that minimize errors and maximize completion of DNA synthesis. Replication fork reversal is one mechanism that helps cells tolerate replication stress. This process involves reannealing of parental template DNA strands and generation of a nascent-nascent DNA duplex. While fork reversal may be beneficial by facilitating DNA repair or template switching, it must be confined to the appropriate contexts to preserve genome stability. Many enzymes have been implicated in this process including ATP-dependent DNA translocases like SMARCAL1, ZRANB3, HLTF, and the helicase FBH1. In addition, the RAD51 recombinase is required. Many additional factors and regulatory activities also act to ensure reversal is beneficial instead of yielding undesirable outcomes. Finally, reversed forks must also be stabilized and often need to be restarted to complete DNA synthesis. Disruption or deregulation of fork reversal causes a variety of human diseases. In this review we will describe the latest models for reversal and key mechanisms of regulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template.

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structurefunction based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways. [ABSTRACT FROM AUTHOR]

In eukaryotes, DNA damage tolerance (DDT) can be achieved through two mechanisms. One mechanism is mediated by the homologous recombination repair proteins. The other is under control of the RAD6 epistatic group genes and is divided into two more pathways, including error-free and error-prone ones. The error-prone mechanism, termed translesion DNA synthesis (TLS), is carried out with the participation of specialized TLS DNA polymerases. TLS is an important source of mutational changes in DNA. On the contrary, upon the realization of RAD6-dependent error-free DDT mechanism, relatively higher accuracy of DNA synthesis is provided by the use of intact sister chromatid or a homologous chromosome as a template to continue replication. In this case, after the replication fork stalling at the site of damage, the 3' end of the synthesized strand is transferred to an intact homologous DNA molecule, the synthesis continues for some length on a new template, and then the elongated strand is transferred back to the original chromatid. Inactivation of most genes that control the error-free DDT mechanism either does not affect the level of UV-induced mutagenesis or decreases it. Exceptions include genes belonging to the HSM3 epistatic group. Mutations in the genes of this group lead to a considerable increase in the frequency of UV-induced mutagenesis. This review focuses on the error-free DDT pathway, and attempts to substantiate the role of the HSM3 epistatic group genes in a series of molecular events that lead to the error-free bypass of replication-blocking lesions in budding yeast are made. [ABSTRACT FROM AUTHOR]

The sliding clamp PCNA is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway. Focusing on Saccharomyces cerevisiae budding yeast, we summarized PCNA related-DDT and repair mechanisms that coordinately sustain genome stability and cell survival. In addition, we compared PCNA sequences from various fungal pathogens, considering recent advances in structural features. Importantly, the identification of PCNA epitopes may lead to potential fungal targets for antifungal drug development.

In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. In this study, we screened a collection of mutations for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress methyl methanesulfonate (MMS) sensitivity of DDT-deficient cells (referred to here as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with decreased MMS-induced mutagenesis relative to that of wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage. [ABSTRACT FROM AUTHOR]

The high‐fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low‐fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading‐strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase–polymerase uncoupling a switch from Pol ε, the canonical leading‐strand replicase, to the lagging‐strand replicase Pol δ, facilitates rapid, efficient and error‐free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8‐oxoguanine. We propose that replicase switching may promote continued leading‐strand synthesis whenever the replisome encounters leading‐strand damage that is bypassed more efficiently by Pol δ than by Pol ε. Synopsis: Complete replication of damaged DNA usually requires dedicated low‐fidelity translesion synthesis polymerases. Here, studies with in vitro‐reconstituted pure yeast replisomes show their inherent ability to bypass the common lesion, thymine glycol, through internal replicase switching. The eukaryotic replisome is tolerant of oxidative base damage in the leading‐strand template.This tolerance does not require canonical translesion synthesis machinery.Base damage causes disengagement of the catalytic subunit of Pol ε from the nascent strand, resulting in uncoupled fork progression.Upon uncoupling, a switch to the lagging strand replicase Pol δ on the nascent leading strand promotes lesion bypass and recoupling.Replicase switching promotes rapid and error‐free lesion bypass at physiological nucleotide levels. [ABSTRACT FROM AUTHOR]

DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome. The Primase and DNA-directed Polymerase (PRIMPOL) is a recently discovered enzyme that possesses both primase and polymerase activities. PRIMPOL is emerging as a key player in DNA damage tolerance, particularly in vertebrate and human cells. Here, we review our current understanding of the function of PRIMPOL in DNA damage tolerance by focusing on the structural aspects that define its dual enzymatic activity, as well as on the mechanisms that control its chromatin recruitment and expression levels. We also focus on the latest findings on the mitochondrial and nuclear functions of PRIMPOL and on the impact of loss of these functions on genome stability and cell survival. Defining the function of PRIMPOL in DNA damage tolerance is becoming increasingly important in the context of human disease. In particular, we discuss recent evidence pointing at the PRIMPOL pathway as a novel molecular target to improve cancer cell response to DNA-damaging chemotherapy and as a predictive parameter to stratify patients in personalized cancer therapy. [ABSTRACT FROM AUTHOR]

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error‐prone, HR uses the sister chromatid and is mostly error‐free. We report that the HR protein Rad52—but not Rad51 and Rad57—acts in concert with the TLS machinery (Rad6/Rad18‐mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light‐induced ssDNA gaps through a non‐recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS‐ and UV‐induced HR); accordingly, Rad52 is required for efficient DNA damage‐induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage‐induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57‐dependent and ‐independent processes, providing a novel role for the recombination proteins in maintaining genome integrity. Synopsis: The recombination protein Rad52 promotes translesion synthesis and mutagenesis by non‐recombinogenic functions, which include the recruitment of the PCNA ubiquitylation complex Rad6/Rad18 to chromatin. The recombination protein Rad52 facilitates the repair of MMS‐and UV‐induced ssDNA lesions through Rad51/Rad57‐dependent and independent non‐recombinogenic functionsRad52, but not Rad51, Rad57and Rad54, is required for efficient DNA damage‐induced mutagenesisRad52, together with Rad51 and Rad57 but not Rad54, facilitates Rad6/Rad18 binding to chromatin and subsequent DNA damage‐induced PCNA ubiquitylation [ABSTRACT FROM AUTHOR]

What is the origin of mutations? In contrast to the naïve notion that mutations are unfortunate accidents, genetic research in microorganisms has demonstrated that most mutations are created by genetically encoded error-prone repair mechanisms. However, error-free repair pathways also exist, and it is still unclear how cells decide when to use one repair method or the other. Here, we summarize what is known about the DNA damage tolerance mechanisms (also known as post-replication repair) for perhaps the best-studied organism, the yeast Saccharomyces cerevisiae. We describe the latest research, which has established the existence of at least two error-free and two error-prone inter-related mechanisms of damage tolerance that compete for the handling of spontaneous DNA damage. We explore what is known about the induction of mutations by DNA damage. We point to potential paradoxes and to open questions that still remain unanswered. [ABSTRACT FROM AUTHOR]

It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sulfolobus islandicus codes for four DNA polymerases: three are of the B-family (Dpo1, Dpo2, and Dpo3), and one is of the Y-family (Dpo4). Western analysis revealed that among the four polymerases, only Dpo2 exhibited DNA damage-inducible expression. To investigate how these DNA polymerases could contribute to DNA damage tolerance in S. islandicus, we conducted genetic analysis of their encoding genes in this archaeon. Plasmid-borne gene expression revealed that Dpo2 increases cell survival upon DNA damage at the expense of mutagenesis. Gene deletion studies showed although dpo1 is essential, the remaining three genes are dispensable. Furthermore, although Dpo4 functions in housekeeping translesion DNA synthesis (TLS), Dpo2, a B-family DNA polymerase once predicted to be inactive, functions as a damage-inducible TLS enzyme solely responsible for targeted mutagenesis, facilitating GC to AT/TA conversions in the process. Together, our data indicate that Dpo2 is the main DNA polymerase responsible for DNA damage tolerance and is the primary source of targeted mutagenesis. Given that crenarchaea encoding a Dpo2 also have a low-GC composition genome, the Dpo2-dependent DNA repair pathway may be conserved in this archaeal lineage.

Most cancers have lost a critical DNA damage response (DDR) pathway during tumor evolution. These alterations provide a useful explanation for the initial sensitivity of tumors to DNA-targeting chemotherapy. A striking example is dysfunctional homology-directed repair (HDR), e.g., due to inactivating mutations in BRCA1 and BRCA2 genes. Extensive efforts are being made to develop novel targeted therapies exploiting such an HDR defect. Inhibitors of poly(ADP-ribose) polymerase (PARP) are an instructive example of this approach. Despite the success of PARP inhibitors, the presence of primary or acquired therapy resistance remains a major challenge in clinical oncology. To move the field of precision medicine forward, we need to understand the precise mechanisms causing therapy resistance. Using preclinical models, various mechanisms underlying chemotherapy resistance have been identified. Restoration of HDR seems to be a prevalent mechanism but this does not explain resistance in all cases. Interestingly, some factors involved in DNA damage response (DDR) have independent functions in replication fork (RF) biology and their loss causes RF instability and therapy sensitivity. However, in BRCA-deficient tumors, loss of these factors leads to restored stability of RFs and acquired drug resistance. In this review we discuss the recent advances in the field of RF biology and its potential implications for chemotherapy response in DDR-defective cancers. Additionally, we review the role of DNA damage tolerance (DDT) pathways in maintenance of genome integrity and their alterations in cancer. Furthermore, we refer to novel tools that, combined with a better understanding of drug resistance mechanisms, may constitute a great advance in personalized diagnosis and therapeutic strategies for patients with HDR-deficient tumors.

The genomes of all living cells are under endogenous and exogenous attacks every day, causing diverse genomic lesions. Most of the lesions can be timely repaired by multiple DNA repair pathways. However, some may persist during S‐phase, block DNA replication, and challenge genome integrity. Eukaryotic cells have evolved DNA damage tolerance (DDT) to mitigate the lethal effects of arrested DNA replication without prior removal of the offending DNA damage. As one important mode of DDT, translesion DNA synthesis (TLS) utilizes multiple low‐fidelity DNA polymerases to incorporate nucleotides opposite DNA lesions to maintain genome integrity. Three different mechanisms have been proposed to regulate the polymerase switching between high‐fidelity DNA polymerases in the replicative machinery and one or more specialized enzymes. Additionally, it is known that proliferating cell nuclear antigen (PCNA) mono‐ubiquitination is essential for optimal TLS. Given its error‐prone property, TLS is closely associated with spontaneous and drug‐induced mutations in cells, which can potentially lead to tumorigenesis and chemotherapy resistance. Therefore, TLS process must be tightly modulated to avoid unwanted mutagenesis. In this review, we will focus on polymerase switching and PCNA mono‐ubiquitination, the two key events in TLS pathway in mammalian cells, and summarize current understandings of regulation of TLS process at the levels of protein–protein interactions, post‐translational modifications as well as transcription and noncoding RNAs. Environ. Mol. Mutagen. 61:680–692, 2020. © 2020 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Sulfolobus islandicus codes for four DNA polymerases: three are of the B-family (Dpo1, Dpo2, and Dpo3), and one is of the Y-family (Dpo4). Western analysis revealed that among the four polymerases, only Dpo2 exhibited DNA damage-inducible expression. To investigate how these DNA polymerases could contribute to DNA damage tolerance in S. islandicus , we conducted genetic analysis of their encoding genes in this archaeon. Plasmid-borne gene expression revealed that Dpo2 increases cell survival upon DNA damage at the expense of mutagenesis. Gene deletion studies showed although dpo1 is essential, the remaining three genes are dispensable. Furthermore, although Dpo4 functions in housekeeping translesion DNA synthesis (TLS), Dpo2, a B-family DNA polymerase once predicted to be inactive, functions as a damage-inducible TLS enzyme solely responsible for targeted mutagenesis, facilitating GC to AT/TA conversions in the process. Together, our data indicate that Dpo2 is the main DNA polymerase responsible for DNA damage tolerance and is the primary source of targeted mutagenesis. Given that crenarchaea encoding a Dpo2 also have a low-GC composition genome, the Dpo2-dependent DNA repair pathway may be conserved in this archaeal lineage. [ABSTRACT FROM AUTHOR]

Most cancers have lost a critical DNA damage response (DDR) pathway during tumor evolution. These alterations provide a useful explanation for the initial sensitivity of tumors to DNA-targeting chemotherapy. A striking example is dysfunctional homology-directed repair (HDR), e.g., due to inactivating mutations in BRCA1 and BRCA2 genes. Extensive efforts are being made to develop novel targeted therapies exploiting such an HDR defect. Inhibitors of poly(ADP-ribose) polymerase (PARP) are an instructive example of this approach. Despite the success of PARP inhibitors, the presence of primary or acquired therapy resistance remains a major challenge in clinical oncology. To move the field of precision medicine forward, we need to understand the precise mechanisms causing therapy resistance. Using preclinical models, various mechanisms underlying chemotherapy resistance have been identified. Restoration of HDR seems to be a prevalent mechanism but this does not explain resistance in all cases. Interestingly, some factors involved in DNA damage response (DDR) have independent functions in replication fork (RF) biology and their loss causes RF instability and therapy sensitivity. However, in BRCA-deficient tumors, loss of these factors leads to restored stability of RFs and acquired drug resistance. In this review we discuss the recent advances in the field of RF biology and its potential implications for chemotherapy response in DDR-defective cancers. Additionally, we review the role of DNA damage tolerance (DDT) pathways in maintenance of genome integrity and their alterations in cancer. Furthermore, we refer to novel tools that, combined with a better understanding of drug resistance mechanisms, may constitute a great advance in personalized diagnosis and therapeutic strategies for patients with HDR-deficient tumors. [ABSTRACT FROM AUTHOR]

TREX2, a 3′-5′ exonuclease, is a part of the DNA damage tolerance (DDT) pathway that stabilizes replication forks (RFs) by ubiquitinating PCNA along with the ubiquitin E3 ligase RAD18 and other DDT factors. Mismatch repair (MMR) corrects DNA polymerase errors, including base mismatches and slippage. Here we demonstrate that TREX2 deletion reduces mutations in cells upon exposure to genotoxins, including those that cause base lesions and DNA polymerase slippage. Importantly, we show that TREX2 generates most of the spontaneous mutations in MMR-mutant cells derived from mice and people. TREX2-induced mutagenesis is dependent on the nuclease and DNA-binding attributes of TREX2. RAD18 deletion also reduces spontaneous mutations in MMR-mutant cells, albeit to a lesser degree. Inactivation of both MMR and TREX2 additively increases RF stalls, while it decreases DNA breaks, consistent with a synthetic phenotype. [Display omitted] • The 3′-5′ exonuclease TREX2 causes spontaneous mutations in cells • TREX2 causes mutations in cells exposed to a range of genotoxins • TREX2 causes spontaneous mutations in MMR-deleted cells • Deletion of TREX2 and MMR has a synthetic effect on DNA replication Mismatch repair (MMR) is a tumor suppressor pathway that suppresses mutations. Marple et al. find that the 3′-5′ exonuclease TREX stabilizes replication forks but also causes spontaneous and genotoxin-stress-induced mutations. In MMR-deleted cells, TREX2 causes nearly all mutations and has an additive defect on replication when co-depleted with MMR. [ABSTRACT FROM AUTHOR]

DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion “i-Damage” system, would elucidate detailed mechanisms governing the choices of specific DDT pathways.

Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη) were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

Replication fork reversal, a critical protective mechanism against replication stress in higher eukaryotic cells, is orchestrated via a series of coordinated enzymatic reactions. The Bloom syndrome gene product, BLM, a member of the highly conserved RecQ helicase family, is implicated in this process, yet its precise regulation and role remain poorly understood. In this study, we demonstrate that the GCFC domain-containing protein TFIP11 forms a complex with the BLM helicase. TFIP11 exhibits a preference for binding to DNA substrates that mimic the structure generated at stalled replication forks. Loss of either TFIP11 or BLM leads to the accumulation of the other protein at stalled forks. This abnormal accumulation, in turn, impairs RAD51-mediated fork reversal and slowing, sensitizes cells to replication stress-inducing agents, and enhances chromosomal instability. These findings reveal a previously unidentified regulatory mechanism that modulates the activities of BLM and RAD51 at stalled forks, thereby impacting genome integrity., (© 2024. The Author(s).)

The Rad5/HLTF protein has a central role in the tolerance to DNA damage by mediating an error-free mode of bypassing unrepaired DNA lesions, and is therefore critical for the maintenance of genome stability. We show in this work that, following cellular stress, Rad5 is regulated by relocalization into two types of nuclear foci that coexist within the same cell, which we termed 'S' and 'I'. Rad5 S-foci form in response to genotoxic stress and are associated with Rad5's function in maintaining genome stability, whereas I-foci form in the presence of proteotoxic stress and are related to Rad5's own proteostasis. Rad5 accumulates into S-foci at DNA damage tolerance sites by liquid-liquid phase separation, while I-foci constitute sites of chaperone-mediated sequestration of Rad5 at the intranuclear quality control compartment (INQ). Relocalization of Rad5 into each type of foci involves different pathways and recruitment mechanisms, but in both cases is driven by the evolutionarily conserved E2 ubiquitin-conjugating enzyme Rad6. This coordinated differential relocalization of Rad5 interconnects DNA damage response and proteostasis networks, highlighting the importance of studying these homeostasis mechanisms in tandem. Spatial regulation of Rad5 under cellular stress conditions thus provides a useful biological model to study cellular homeostasis as a whole., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.

Completion of DNA replication relies on the ability of replication forks to traverse various types of DNA damage, actively transcribed regions, and structured DNA. The mechanisms enabling these processes are here referred to as DNA damage tolerance pathways. Here, we depict the stalled DNA replication fork structures with main DNA transactions and key factors contributing to the bypass of such blocks, replication restart, and completion. To view this SnapShot, open or download the PDF., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cell-free extracts derived from Xenopus eggs have been widely used to decipher molecular pathways involved in several cellular processes including DNA synthesis, the DNA damage response, and genome integrity maintenance. We set out assays using Xenopus cell-free extracts to study translesion DNA synthesis (TLS), a branch of the DNA damage tolerance pathway that allows replication of damaged DNA. Using this system, we were able to recapitulate TLS activities that occur naturally in vivo during early embryogenesis. This chapter describes protocols to detect chromatin-bound TLS factors by western blotting and immunofluorescence microscopy upon induction of DNA damage by UV irradiation, monitor TLS-dependent mutagenesis, and perform proteomic screening., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA is constantly exposed to a wide variety of exogenous and endogenous agents, and most DNA lesions inhibit DNA synthesis. To cope with such problems during replication, cells have molecular mechanisms to resume DNA synthesis in the presence of DNA lesions, which are known as DNA damage tolerance (DDT) pathways. The concept of ubiquitination-mediated regulation of DDT pathways in eukaryotes was established via genetic studies in the yeast Saccharomyces cerevisiae, in which two branches of the DDT pathway are regulated via ubiquitination of proliferating cell nuclear antigen (PCNA): translesion DNA synthesis (TLS) and homology-dependent repair (HDR), which are stimulated by mono- and polyubiquitination of PCNA, respectively. Over the subsequent nearly two decades, significant progress has been made in understanding the mechanisms that regulate DDT pathways in other eukaryotes. Importantly, TLS is intrinsically error-prone because of the miscoding nature of most damaged nucleotides and inaccurate replication of undamaged templates by TLS polymerases (pols), whereas HDR is theoretically error-free because the DNA synthesis is thought to be predominantly performed by pol δ, an accurate replicative DNA pol, using the undamaged sister chromatid as its template. Thus, the regulation of the choice between the TLS and HDR pathways is critical to determine the appropriate biological outcomes caused by DNA damage. In this review, we summarize our current understanding of the species-specific regulatory mechanisms of PCNA ubiquitination and how cells choose between TLS and HDR. We then provide a hypothetical model for the spatiotemporal regulation of DDT pathways in human cells. [ABSTRACT FROM AUTHOR]

The eukaryotic post-replication repair (PRR) pathway allows completion of DNA replication when replication forks encounter lesions on the DNA template and are mediated by post-translational ubiquitination of the DNA sliding clamp proliferating cell nuclear antigen (PCNA). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases to replicate past DNA lesions in an error-prone manner while addition of K63-linked polyubiquitin chains signals for error-free template switching to the sister chromatid. Central to both branches is the E3 ubiquitin ligase and DNA helicase Rad5/helicase-like transcription factor (HLTF). Mutations in PRR pathway components lead to genomic rearrangements, cancer predisposition, and cancer progression. Recent studies have challenged the notion that the PRR pathway is involved only in DNA lesion tolerance and have shed new light on its roles in cancer progression. Molecular details of Rad5/HLTF recruitment and function at replication forks have emerged. Mounting evidence indicates that PRR is required during lesion-less replication stress, leading to TLS polymerase activity on undamaged templates. Analysis of PRR mutation status in human cancers and PRR function in cancer models indicates that down regulation of PRR activity is a viable strategy to inhibit cancer cell growth and reduce chemoresistance. Here, we review these findings, discuss how they change our views of current PRR models, and look forward to targeting the PRR pathway in the clinic. [ABSTRACT FROM AUTHOR]