Completion Of DNA Replication Research Articles

Abstract A035: Checkpoint is the control mechanism to ensure the strict order of cellular events during cell cycle. Here, we show that cells enter mitosis without completing DNA replication although with transient activation of known ATR/Chk1 checkpoint

Abstract Checkpoints are the control mechanisms that ensure the events of the cell cycle strictly ordered into dependent pathways. Notably, genome duplication and chromosome segregation are two major events of every cell cycle, which respectively occur in S phase and mitosis (M). Checkpoint either for the S/G2 or G2/M transition is considered of great importance to ensure the completion of DNA replication before the initiation of mitosis. Moreover, the degradation tag (dTAG) system allows precise and well-controlled analysis of essential genes by inducing immediate degradation of the protein of interest in a strictly defined time window such as a specific cell cycle phase. To answer what if cells cannot complete DNA replication in mammalian cells, we employed dTAG system targeting DNA replication machinery and generated homozygous dTAG C-terminal knockin at the CDC45/MCM2/GINS4 (CMG) locus in HEK293A cells. Our data show that the CDC45/MCM2/GINS4-dTAG-HA proteins as well as DNA replication rapidly decreased to significantly lower levels in the presence of dTAGv-1 only for 1 hour than that in cells treated with dTAG-NEG. Contrary to the textbook model, our data show that the HEK293A-CMG-dTAG cells treated with dTAGv-1 for 24 to 48 hours, which did not complete DNA replication, began to die instead of being arrested. Moreover, although we observed the transient ATR/Chk1 checkpoint activation when the HEK293A-CMG-dTAG cells treated with dTAGv-1 within 24 hours, the ATR/Chk1 checkpoint disappeared but followed by increased CyclinB1 and pH3S10 protein levels after 24 hours. These data imply that the completion of DNA replication is not under the surveillance of known ATR/Chk1 checkpoint. We then hypothesized that CMG-depleted cells could enter and die in mitosis regarding the increased levels of CyclinB1. To test this, we first transduced HEK293A-CMG-dTAG cells with the cell cycle biosensor, Fucci (CA). HEK293A-CMG-dTAG-Fucci (CA) cells were synchronized in S phase followed by dTAG-NEG or dTAGv-1 treatment. DTAG-NEG-treated cells divided repeatedly, while dTAGv-1-treated cells entered in G2 or M phase without cell division as well as achieving 4N DNA content. To address in which precise phase of CMG-depleted cells die, G2 or M phase, we also transduced HEK293A-CMG-dTAG cells with GFP-H2B. By synchronizing cells in S phase, we observed that dTAGv-1-treated cells either exhibited condensed DNA that occurring in early mitosis or failed in chromosome segregation at later M, suggesting that these cells entered and died in mitosis. Our data demonstrate that the completion of DNA replication is not the prerequisite for cells to enter mitosis in CMG-depleted cells, although with transient activation of the intact ATR/Chk1 checkpoint. However, it is possible that CMG complex is the sensor for monitoring the accomplishment of genome duplication. Therefore, it is necessary to generate HEK293A-PCNA/RPA(1-3)-dTAG cells to test whether PCNA/RPA-depleted cells could recapitulate the phenotypes of CMG-depleted cells. Citation Format: Min Huang, Junjie Chen. Checkpoint is the control mechanism to ensure the strict order of cellular events during cell cycle. Here, we show that cells enter mitosis without completing DNA replication although with transient activation of known ATR/Chk1 checkpoint [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A035.

MutSβ protects common fragile sites by facilitating homology-directed repair at DNA double-strand breaks with secondary structures.

Common fragile sites (CFSs) are regions prone to chromosomal rearrangements, thereby contributing to tumorigenesis. Under replication stress (RS), CFSs often harbor under-replicated DNA regions at the onset of mitosis, triggering homology-directed repair known as mitotic DNA synthesis (MiDAS) to complete DNA replication. In this study, we identified an important role of DNA mismatch repair protein MutSβ (MSH2/MSH3) in facilitating MiDAS and maintaining CFS stability. Specifically, we demonstrated that MutSβ is required for the increased mitotic recombination induced by RS or FANCM loss at CFS-derived AT-rich and structure-prone sequences (CFS-ATs). We also found that MSH3 exhibits synthetic lethality with FANCM. Mechanistically, MutSβ is required for homologous recombination (HR) especially when DNA double-strand break (DSB) ends contain secondary structures. We also showed that upon RS, MutSβ is recruited to Flex1, a specific CFS-AT, in a PCNA-dependent but MUS81-independent manner. Furthermore, MutSβ interacts with RAD52 and promotes RAD52 recruitment to Flex1 following MUS81-dependent fork cleavage. RAD52, in turn, recruits XPF/ERCC1 to remove DNA secondary structures at DSB ends, enabling HR/break-induced replication (BIR) at CFS-ATs. We propose that the specific requirement of MutSβ in processing DNA secondary structures at CFS-ATs underlies its crucial role in promoting MiDAS and maintaining CFS integrity.

Lnk/Sh2b3 Deficiency Increases DNA Damage Tolerance to Promote Hematopoietic Stem Cell Fitness

DNA repair deficiency depicted by the syndrome Fanconi Anemia (FA), results in progressive bone marrow failure (BMF), increased leukemia incidence and cancer susceptibility. Out of the 23 causative genes for FA, FANCD2 is crucial for its central role in the regulation of DNA repair and replication. The FA pathway plays an important role in maintaining hematopoietic stem and progenitor cell (HSPC) homeostasis and genome integrity. Our previous studies revealed that loss of the adaptor protein LNK (SH2B3), a negative regulator of cytokine signaling, restores hematopoietic stem cell (HSC) function and genome integrity in Fancd2 deficient FA mouse models and primary human FA progenitors. Notably, mice deficient in Lnk harbor a remarkable 14-fold expansion of HSCs with superior self-renewal capability. However, the protective mechanisms that LNK controls to prevent stem cell attrition and increase stem cell fitness are not established. Here, we show that Lnk deficiency reduced DNA damage in Fancd2-/- HSCs at the steady-state in vivo, as well as in forced proliferation upon transplantation. Lnk deficiency reduced HSPC DNA damage in replication stress upon cytokine-induced proliferation in ex vivo culture, accompanied by reduced p53 activation. One injection of pIpC induces HSPC into the cell cycle within 24hr, thus we used this as a model to study replication stress in vivo. We found that Lnk deficiency reduced ATR but not ATM pathway activation in Fancd2-/- HSCs in pIpC-induced proliferation in vivo. ATR is activated by single-strand DNA breaks (SSBs) during replication stress; thus we directly examined the amount of single-strand DNA (ssDNA) and the level of ssDNA binding protein RPA (Replication Protein A) in HSCs during the cell cycle. Of note, Lnk deficiency reduced ssDNA as well as chromatin-bound RPA2 in Fancd2-/- HSCs in replication in vivo. Lnk deficiency also reduced RPA2 phosphorylation in HSPCs under Camptothecin (CPT) and hydroxyurea (HU) induced replication stress conditions. Mechanistically, loss of Lnk protected stalled replication fork from degradation, suppressed ssDNA gaps, and promoted replication fork restart in Fancd2-/- HSPCs upon HU-induced replication stress. In light of our data suggesting a new role for LNK in replication stress-induced DNA damage, we examined the DNA damage tolerance (DDT) mechanism that enables DNA replication to circumvent the lesions, thereby allowing the completion of DNA replication, increasing the survival of HSPCs and preventing genome instability. One of the major DDT pathways is translesion synthesis (TLS) in which the replicative DNA polymerase is temporarily replaced by special TLS polymerases pol ζ or η that can replicate across DNA lesions. Indeed, we found that Lnk deficient HSCs have increased chromatin-bound TLS polymerase eta (Pol ƞ) and were resistant to shRNA-mediated knockdown of PolH (the gene encodes polymerase eta) compared to wildtype HSCs in transplant assays. Strikingly, Lnk deficient HSCs were more resistant to the inhibitor of Rev1 that disrupts Rev1/ pol ζ interaction than wildtype HSCs in transplanting assays. These findings highlight a previously-unappreciated role of LNK in limiting DNA damage tolerance mechanisms in both normal and diseased replicative states. The novel link between LNK-controlled cytokine signaling and TLS sheds light on our understanding of replication stress mitigation to promote HSC fitness that can be explored for future regeneration medicine.

The Adaptive Mechanisms and Checkpoint Responses to a Stressed DNA Replication Fork

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.

Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli.

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species. IMPORTANCE Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions in vivo. In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.

Chi sequences switch the RecBCD helicase–nuclease complex from degradative to replicative modes during the completion of DNA replication

Accurately completing DNA replication when two forks converge is essential to genomic stability. The RecBCD helicase-nuclease complex plays a central role in completion by promoting resection and joining of the excess DNA created when replisomes converge. chi sequences alter RecBCD activity and localize with cross-over hotspots during sexual events in bacteria, yet their functional role during chromosome replication remains unknown. Here, we use two-dimensional agarose gel analysis to show that chi induces replication on substrates containing convergent forks. The induced-replication is processive, but uncoupled with respect to leading and lagging strand synthesis, and can be suppressed by ter sites which limit replisome progression. Our observations demonstrate that convergent replisomes create a substrate that is processed by RecBCD, and that chi, when encountered, switches RecBCD from a degradative to replicative function. We propose that chi serves to functionally differentiate DNA ends created during completion, which require degradation, from those created by chromosomal double-strand breaks, which require resynthesis.

Role of Cockayne Syndrome Group B Protein in Replication Stress: Implications for Cancer Therapy.

A variety of endogenous and exogenous insults are capable of impeding replication fork progression, leading to replication stress. Several SNF2 fork remodelers have been shown to play critical roles in resolving this replication stress, utilizing different pathways dependent upon the nature of the DNA lesion, location on the DNA, and the stage of the cell cycle, to complete DNA replication in a manner preserving genetic integrity. Under certain conditions, however, the attempted repair may lead to additional genetic instability. Cockayne syndrome group B (CSB) protein, a SNF2 chromatin remodeler best known for its role in transcription-coupled nucleotide excision repair, has recently been shown to catalyze fork reversal, a pathway that can provide stability of stalled forks and allow resumption of DNA synthesis without chromosome breakage. Prolonged stalling of replication forks may collapse to give rise to DNA double-strand breaks, which are preferentially repaired by homology-directed recombination. CSB plays a role in repairing collapsed forks by promoting break-induced replication in S phase and early mitosis. In this review, we discuss roles of CSB in regulating the sources of replication stress, replication stress response, as well as the implications of CSB for cancer therapy.

Reconstitution of a telomeric replicon organized by CST

Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. This replication requires telomerase3 extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)n); the synthesis of the complementary C-strand ((CCCTAA)n) is much less well characterized. The CST (CTC1–STN1–TEN1) protein complex, a DNA polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6–9, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST–Polα–primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3′ overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα–primase to telomeric ssDNA4,12,13 but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.

DNA Sequence Specificity Reveals a Role of the HLTF HIRAN Domain in the Recognition of Trinucleotide Repeats.

DNA damage tolerance (DDT) pathways enable cells to cope with a variety of replication blocks that threaten their ability to complete DNA replication. Helicase-like transcription factor (HLTF) plays a central role in the error-free DDT pathway, template switching (TS), by serving as a ubiquitin ligase to polyubiquitinate the DNA sliding clamp PCNA, which promotes TS initiation. HLTF also serves as an ATP-dependent DNA translocase facilitating replication fork remodeling. The HIP116, Rad5p N-terminal (HIRAN) domain of HLTF specifically recognizes the unmodified 3'-end of single-stranded DNA (ssDNA) at stalled replication forks to promote fork regression. Several crystal structures of the HIRAN domain in complex with ssDNA have been reported; however, optimal ssDNA sequences for high-affinity binding with the domain have not been described. Here we elucidated DNA sequence preferences of HLTF HIRAN through systematic studies of its binding to ssDNA substrates using fluorescence polarization assays and a computational analysis of the ssDNA:HIRAN interaction. These studies reveal that the HLTF HIRAN domain preferentially recognizes a (T/C)TG sequence at the 3'-hydroxyl ssDNA end, which occurs in the CTG trinucleotide repeat (TNR) regions that are susceptible to expansion and deletion mutations identified in neuromuscular and neurodegenerative disorders. These findings support a role for HLTF in maintaining the stability of difficult to replicate TNR microsatellite regions.

CDC7 kinase (DDK) inhibition disrupts DNA replication leading to mitotic catastrophe in Ewing sarcoma

Ewing sarcoma is the second most common bone malignancy in children and adolescents. In recent years, a large body of evidence has emerged that suggests Ewing tumors harbor large amounts of replication stress (RS). CDC7, also known as DDK (DBF4-dependent kinase), is a serine/threonine kinase that is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the RS response. Due to DDK’s diverse roles during replication, coupled with the fact that there is an increased level of RS within Ewing tumors, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibition. Here, we report that DDK inhibition resulted a significant reduction in cell viability and the induction of apoptosis, specifically in Ewing sarcoma cells. Treatment with DDK inhibitors dramatically reduced the rate of replication, prolonged S-phase, and led to a pronounced increase in phospho-CDC2 (Y15), indicating delay of mitotic entry. The induction of cell death corresponded to mitotic exit and G1 entry, suggesting improper mitotic progression. In accordance with this, we find that DDK inhibition caused premature mitotic entry resulting in mitotic abnormalities such as anaphase bridges, lagging chromosomes, and cells with >2 poles in Ewing sarcoma cells. This abnormal progression through mitosis resulted in mitotic catastrophe as evidenced by the formation of micronuclei and induction of DNA damage. Together, these findings suggest that DDK activity is required for the faithful and timely completion of DNA replication in Ewing cells and that DDK inhibition may present a viable therapeutic strategy for the treatment of Ewing sarcoma.

Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.

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

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.

CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal.

CDK4/6 inhibitors arrest the cell cycle in G1‐phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long‐lasting effects on tumour growth. Here, we demonstrate that a prolonged G1 arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. Upon release from that arrest, many cells fail to complete DNA replication and exit the cell cycle in a p53‐dependent manner. If cells fail to withdraw from the cell cycle following DNA replication problems, they enter mitosis and missegregate chromosomes causing excessive DNA damage, which further limits their proliferative potential. These effects are observed in a range of tumour types, including breast cancer, implying that genotoxic stress is a common outcome of CDK4/6 inhibition. This unanticipated ability of CDK4/6 inhibitors to induce DNA damage now provides a rationale to better predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

DNA replication: the recombination connection.

Failure to complete DNA replication is one of the major sources of genome instability leading to aneuploidy, chromosome breakage, and chromosome rearrangements that are associated with human cancer. One of the surprising revelations of the past decade is that the completion of replication at so-called common fragile sites (CFS) occurs very late in the cell cycle - at mitosis - through a process termed MiDAS (mitotic DNA synthesis). MiDAS is strongly related to another cancer-promoting phenomenon: the activation of alternative lengthening of telomeres (ALT). Our understanding of the mechanisms of ALT and MiDAS in mammalian cells has drawn heavily from recent advances in the study of break-induced replication (BIR), especially in budding yeast. We provide new insights into the BIR, MiDAS, and ALT pathways and their shared similarities.

Microscopic Detection of DNA Synthesis in Early Mitosis at Repetitive lacO Sequences in Human Cells.

In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork.

SummaryThe AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.

Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance.

The DNA damage tolerance (DDT) response is aimed to timely and safely complete DNA replication by facilitating the advance of replication forks through blocking lesions. This process is associated with an accumulation of single-strand DNA (ssDNA), both at the fork and behind the fork. Lesion bypass and ssDNA filling can be performed by translation synthesis (TLS) and template switching mechanisms. TLS uses low-fidelity polymerases to incorporate a dNTP opposite the blocking lesion, whereas template switching uses a Rad51/ssDNA nucleofilament and the sister chromatid to bypass the lesion. Rad51 is loaded at this nucleofilament by two mediator proteins, BRCA2 and Rad52, and these three factors are critical for homologous recombination (HR). Here, we review recent advances showing that Rad51, BRCA2, and Rad52 perform some of these functions through mechanisms that do not require the strand exchange activity of Rad51: the formation and protection of reversed fork structures aimed to bypass blocking lesions, and the promotion of TLS. These findings point to the central HR proteins as potential molecular switches in the choice of the mechanism of DDT.

Efficiency and equity in origin licensing to ensure complete DNA replication.

The cell division cycle must be strictly regulated during both development and adult maintenance, and efficient and well-controlled DNA replication is a key event in the cell cycle. DNA replication origins are prepared in G1 phase of the cell cycle in a process known as origin licensing which is essential for DNA replication initiation in the subsequent S phase. Appropriate origin licensing includes: (1) Licensing enough origins at adequate origin licensing speed to complete licensing before G1 phase ends; (2) Licensing origins such that they are well-distributed on all chromosomes. Both aspects of licensing are critical for replication efficiency and accuracy. In this minireview, we will discuss recent advances in defining how origin licensing speed and distribution are critical to ensure DNA replication completion and genome stability.

Mind the replication gap.

Unlike bacteria, mammalian cells need to complete DNA replication before segregating their chromosomes for the maintenance of genome integrity. Thus, cells have evolved efficient pathways to restore stalled and/or collapsed replication forks during S-phase, and when necessary, also to delay cell cycle progression to ensure replication completion. However, strong evidence shows that cells can proceed to mitosis with incompletely replicated DNA when under mild replication stress (RS) conditions. Consequently, the incompletely replicated genomic gaps form, predominantly at common fragile site regions, where the converging fork-like DNA structures accumulate. These branched structures pose a severe threat to the faithful disjunction of chromosomes as they physically interlink the partially duplicated sister chromatids. In this review, we provide an overview discussing how cells respond and deal with the under-replicated DNA structures that escape from the S/G2 surveillance system. We also focus on recent research of a mitotic break-induced replication pathway (also known as mitotic DNA repair synthesis), which has been proposed to operate during prophase in an attempt to finish DNA synthesis at the under-replicated genomic regions. Finally, we discuss recent data on how mild RS may cause chromosome instability and mutations that accelerate cancer genome evolution.

DNA Breaks Due to Replication Stress Limit the Developmental Potential of Human Preimplantation Embryos

Human cleavage stage embryos frequently acquire chromosomal aneuploidies during mitosis. We recently showed that double strand breaks (DSBs) introduced by Cas9 result in frequent chromosomal alterations. Here we show that spontaneous DSBs arise de novo during embryonic DNA replication and show as chromosomal breaks that occur primarily, in centromeric, and gene-poor areas, with the latter harboring long genes implicated in neurodevelopmental disorders, including CNTN5, IMMP2L, LRP1B, RYR2, CSMD1. While transcription-replication conflicts are not required for chromosome fragility, fragile, gene-poor regions contain long-traveling replication forks, and complete replication late in G2 phase, hours after most of the genome has been duplicated. Incomplete replication at these sites in both human and mouse zygotes, results in DSBs, whole and segmental chromosome errors, micronucleation, chromosome fragmentation and poor embryo quality. Thus, failure to complete DNA replication is a mechanism of developmental failure, and a source of detrimental genetic alterations in humans.