The coupling of epigenome replication with DNA replication

In multicellular organisms, each cell contains the same DNA sequence, but with different epigenetic information that determines the cell specificity. Semi-conservative DNA replication faithfully copies the parental nucleotide sequence into two DNA daughter strands during each cell cycle. At the same time, epigenetic marks such as DNA methylation and histone modifications are either precisely transmitted to the daughter cells or dynamically changed during S-phase. Recent studies indicate that in each cell cycle, many DNA replication related proteins are involved in not only genomic but also epigenomic replication. Histone modification proteins, chromatin remodeling proteins, histone variants, and RNAs participate in the epigenomic replication during S-phase. As a consequence, epigenome replication is closely linked with DNA replication during S-phase.     

Regulation of DNA synthesis in bacteria: Analysis of the Bates/Kleckner licensing/initiation-mass model for cell cycle control

Bates and Kleckner have recently proposed that bacterial cell division is a licensing agent for a subsequent initiation of DNA replication. They also propose that initiation mass for DNA replication is not constant. These two proposals do not take into account older data showing that initiation of DNA replication can occur prior to the division event. This critical analysis is derived from measurements of DNA replication during the division cycle in cells growing at different, and more rapid, growth rates. Furthermore, mutants impaired in division can initiate DNA synthesis. The data presented by Bates and Kleckner do not support the proposal that initiation mass is variable, and the proposed pattern of DNA replication during the division cycle of the K12 cells analysed is not consistent with prior data on the pattern of DNA replication during the division cycle.     

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

Summary The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 μM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 μM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.     

The S-phase checkpoint and its regulation in Saccharomyces cerevisiae

Cells are never more vulnerable than during DNA replication, which represents a major moment of potential genetic instability. Genotoxic insults induce many different forms of DNA damage that may interfere with the ability of cells to properly duplicate their genome. Primary damage may in turn undergo structural transformations during DNA replication, thus generating secondary lesions that may be even more dangerous. Cells experiencing replication of damaged DNA or replication blocks activate an S-phase checkpoint response that assures the fidelity and completion of DNA replication before cells enter M-phase. The S-phase checkpoint pathway regulates not only progress through the cell cycle but also DNA repair and DNA replication itself.     

Fanconi anemia proteins stabilize replication forks

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.     

Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts

The homotrimeric DNA replication protein proliferating cell nuclear antigen (PCNA) is regulated by both ubiquitylation and sumoylation. We study the appearance and the impact of these modifications on chromosomal replication in frog egg extracts. Xenopus laevis PCNA is modified on lysine 164 by sumoylation, monoubiquitylation, and diubiquitylation. Sumoylation and monoubiquitylation occur during the replication of undamaged DNA, whereas diubiquitylation occurs specifically in response to DNA damage. When lysine 164 modification is prevented, replication fork movement through undamaged DNA slows down and DNA polymerase delta fails to associate with replicating chromatin. When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates. Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA. Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.     

Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damaged.     

Herpes Simplex Virus 1 DNA Is in Unstable Nucleosomes throughout the Lytic Infection Cycle,and the Instability of the Nucleosomes Is Independent of DNA Replication

Herpes simplex virus 1 (HSV-1) DNA is chromatinized during latency and consequently regularly digested by micrococcal nuclease (MCN) to nucleosome-size fragments. In contrast, MCN digests HSV-1 DNA in lytically infected cells to mostly heterogeneous sizes. Yet HSV-1 DNA coimmunoprecipitates with histones during lytic infections. We have shown that at 5 h postinfection, most nuclear HSV-1 DNA is in particularly unstable nucleoprotein complexes and consequently is more accessible to MCN than DNA in cellular chromatin. HSV-1 DNA was quantitatively recovered at this time in complexes with the biophysical properties of mono- to polynucleosomes following a modified MCN digestion developed to detect potential unstable intermediates. We proposed that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infections. Physiologically, nucleosome assembly typically associates with DNA replication, although DNA replication transiently disrupts nucleosomes. It therefore remained unclear whether the instability of the HSV-1 nucleoprotein complexes was related to the ongoing viral DNA replication. Here we tested whether HSV-1 DNA is in unstable nucleosome-like complexes before, during, or after the peak of viral DNA replication or when HSV-1 DNA replication is inhibited. HSV-1 DNA was quantitatively recovered in complexes fractionating as mono- to polynucleosomes from nuclei harvested at 2, 5, 7, or 9 h after infection, even if viral DNA replication was inhibited. Therefore, most HSV-1 DNA is in unstable nucleosome-like complexes throughout the lytic replication cycle, and the instability of these complexes is surprisingly independent of HSV-1 DNA replication. The specific accessibility of nuclear HSV-1 DNA, however, varied at different times after infection.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork

Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundred fold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decrease following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. Furthermore, we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE-GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE-GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNA replication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.     

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.     

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF^Pof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.     

Replication fork dynamics and the DNA damage response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.     

In vivo accumulation of cyclin A and cellular replication factors in autonomous parvovirus minute virus of mice-associated replication bodies

Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellular factors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds in specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases alpha and delta in these bodies suggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruitment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement in activating the conversion of virion single-stranded DNA to the duplex replicative form.     

The functional role of Cdc6 in S-G2/M in mammalian cells

The Cdc6 protein is required for licensing of replication origins before the onset of DNA replication in eukaryotic cells. Here, we examined whether Cdc6 has other roles in mammalian cell-cycle progression from S to G2/M phase. Using RNA interference, we showed that depletion of Cdc6 in synchronous G1 cells blocks G1 to S transition, confirming the essential role of Cdc6 in the initiation of DNA replication. In contrast, depletion of Cdc6 in synchronous S-phase cells slowed DNA replication and led to mitotic lethality. The Cdc6-depleted S-phase cells showed fewer newly fired origins; however, established replication forks remained active, even during chromatin condensation. Despite such DNA replication abnormalities, loss of Cdc6 failed to activate Chk1 kinase. These results show that Cdc6 is not only required for G1 origin licensing, but is also crucial for proper S-phase DNA replication that is essential for DNA segregation during mitosis.     

Regulation of DNA replication during the cell cycle: roles of Cdc7 kinase and coupling of replication, recombination, and repair in response to replication fork arrest

DNA replication is central to cell growth, development, and generation of tissues and organs. Recent advances in understanding replication machinery have revealed striking conservation of components involved in the processes of DNA replication, from yeasts to human. The conservation extends even to bacteria for some basic components of replication apparatus. Eukaryotic DNA replication is regulated at various stages to ensure strict regulation during cell cycle. We have identified a novel mammalian kinase, Cdc7-ASK (Activator of S phase Kinase), that plays a key role at the entry into S phase as a molecular switch for DNA replication. This kinase is specifically activated during S phase and triggers the firing of DNA replication by phosphorylating an essential DNA helicase component of the replication complex. Environmental stresses such as DNA damages or depletion of essential nutrients for DNA synthesis lead to unscheduled arrest of DNA replication forks. In bacteria, this leads to induction of altered modes of DNA replication, which may repair DNA damages, facilitate reassembly of replication machinery at the stalled replication fork, or do both. In eukaryotes, blocking replication forks usually induces both checkpoint responses, which prevent premature progression of cell cycle events before precise completion of the preceding cell cycle stage, and the recombinational repair system for the lesions. Possible common bases in recognition of stalled replication forks in bacteria and eukaryotes will be discussed. Furthermore, we will discuss the potential of replication and checkpoint proteins as targets of anticancer agents as well as possible novel technology for stem cell amplification through manipulation of DNA replication.     

Molecular Weight of Deoxyribonucleic Acid Synthesized During Initiation of Chromosome Replication in Escherichia coli

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.     

In vitro reconstitution of the end replication problem

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.     

The DNA damage response during an unperturbed S-phase

DNA replication is a highly conserved and controlled process. To maintain genome integrity, the DNA must be faithfully duplicated once before chromosomes are segregated to daughter cells. Experimental insults to cells during DNA replication trigger an array of responses to help cells cope with DNA damage and replication stress. This has been coined the DNA damage response. During an unperturbed S-phase, DNA lesions and aberrant DNA structures arise as a consequence of normal DNA replication. Recent data suggest that the same pathways regulating the response to acute DNA damage also operate during normal S-phase to maintain genome integrity in the face of low levels of damage. This review will focus on the role of key proteins and signaling pathways, originally identified by their requirement to maintain genome stability during DNA replication following experimental insults, in the regulation of progression through normal S-phase.