Completion Of Chromosome Replication Research Articles

Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.

Preparation for DNA replication: the key to a successful S phase.

Successful genome duplication is required for cell proliferation and demands extraordinary precision and accuracy. The mechanisms by which cells enter, progress through, and exit S phase are intense areas of focus in the cell cycle and genome stability fields. Key molecular events in the G1 phase of the cell division cycle, especially origin licensing, are essential for pre-establishing conditions for efficient DNA replication during the subsequent S phase. If G1 events are poorly regulated or disordered, then DNA replication can be compromised leading to genome instability, a hallmark of tumorigenesis. Upon entry into S phase, coordinated origin firing and replication progression ensure complete, timely, and precise chromosome replication. Both G1 and S phase progressions are controlled by master cell cycle protein kinases and ubiquitin ligases that govern the activity and abundance of DNA replication factors. In this short review, we describe current understanding and recent developments related to G1 progression and S phase entrance and exit with a particular focus on origin licensing regulation in vertebrates.

The Initial Response of a Eukaryotic Replisome to DNA Damage

SummaryThe replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome’s initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins.

SbcC-SbcD and ExoI process convergent forks to complete chromosome replication

SbcC-SbcD are the bacterial orthologs of Mre11-Rad50, a nuclease complex essential for genome stability, normal development, and viability in mammals. In vitro, these enzymes degrade long DNA palindromic structures. When inactivated along with ExoI in Escherichia coli, or Sae2 in eukaryotes, palindromic amplifications arise and propagate in cells. However, long DNA palindromes are not normally found in bacterial or human genomes, leaving the cellular substrates and function of these enzymes unknown. Here, we show that during the completion of DNA replication, convergent replication forks form a palindrome-like structural intermediate that requires nucleolytic processing by SbcC-SbcD and ExoI before chromosome replication can be completed. Inactivation of these nucleases prevents completion from occurring, and under these conditions, cells maintain viability by shunting the reaction through an aberrant recombinational pathway that leads to amplifications and instability in this region. The results identify replication completion as an event critical to maintain genome integrity and cell viability, demonstrate SbcC-SbcD-ExoI acts before RecBCD and is required to initiate the completion reaction, and reveal how defects in completion result in genomic instability.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways—DNA replication and ribosome biogenesis.

RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms.

Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process - after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double-strand breaks and replication fork collapse occur far less frequently than previously speculated.

Determining if Telomeres Matter in Colon Cancer Initiation or Progression

The nucleus of each human cell contains 46 linear chromosomes (92 ends) that are capped by thousands of repetitive TTAGGG DNA sequences (similar to the plastic tips on shoelaces). These ends, called “telomeres” have complex roles in aging and cancer and have proven to be intricately involved in such pivotal processes as the protection of genetic material, the completion of chromosome replication, the regulation of cellular aging due to progressively shortening telomeres throughout life, the initial protection against unlimited cellular growth, and, in combination with other alterations, the promotion of cancer progression. A collection of six proteins that either bind or associate with telomeres (the shelterin complex) begin to resolve a central question of how cells distinguish telomere ends from typical genomic DNA double-strand breaks (1). The consequences of telomere dysfunction are becoming more apparent by examining a growing list of human genetic diseases called telomeropathies. In certain inherited and familial cases of idiopathic pulmonary fibrosis, dyskeratosis congenita, and sporadic bone marrow failure (aplastic anemia), inheritance of short telomeres due to mutations in genes involved in the ribonucleoprotein telomerase holoenzyme lead to premature aging phenotypes (2). Telomerase is the cellular reverse transcriptase that can add TTAGGG repeats onto the telomeres and is active during early human development (3) but is silent in most adult tissues except proliferating stem cells, unless it is upregulated as part of cancer progression (4).

Overlap of replication rounds disturbs the progression of replicating forks in a ribonucleotide reductase mutant of Escherichia coli

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, Escherichia coli cells carrying the nrdA101 allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 °C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 °C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 °C cannot explain the completion of chromosomal DNA replication in the nrdA101 mutant. We found that inactivation of the DnaA protein by using several dnaAts alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the nrdA101 mutant at 42 °C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies of datA sequence increases the relative amount of DNA synthesis of the nrdA101 mutant at 42 °C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.

TopBP1 Deficiency Causes an Early Embryonic Lethality and Induces Cellular Senescence in Primary Cells

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.

The DNA replication checkpoint aids survival of plants deficient in the novel replisome factor ETG1

Complete and accurate chromosomal DNA replication is essential for the maintenance of the genetic integrity of all organisms. Errors in replication are buffered by the activation of DNA stress checkpoints; however, in plants, the relative importance of a coordinated induction of DNA repair and cell cycle-arresting genes in the survival of replication mutants is unknown. In a systematic screen for Arabidopsis thaliana E2F target genes, the E2F TARGET GENE 1 (ETG1) was identified as a novel evolutionarily conserved replisome factor. ETG1 was associated with the minichromosome maintenance complex and was crucial for efficient DNA replication. Plants lacking the ETG1 gene had serrated leaves due to cell cycle inhibition triggered by the DNA replication checkpoints, as shown by the transcriptional induction of DNA stress checkpoint genes. The importance of checkpoint activation was highlighted by double mutant analysis: whereas etg1 mutant plants developed relatively normally, a synthetically lethal interaction was observed between etg1 and the checkpoint mutants wee1 and atr, demonstrating that activation of a G2 cell cycle checkpoint accounts for survival of ETG1-deficient plants.

The Role of Stn1p inSaccharomyces cerevisiaeTelomere Capping Can Be Separated From Its Interaction With Cdc13p

The function of telomeres is twofold: to facilitate complete chromosome replication and to protect chromosome ends against fusions and illegitimate recombination. In the budding yeast Saccharomyces cerevisiae, interactions among Cdc13p, Stn1p, and Ten1p are thought to be critical for promoting these processes. We have identified distinct Stn1p domains that mediate interaction with either Ten1p or Cdc13p, allowing analysis of whether the interaction between Cdc13p and Stn1p is indeed essential for telomere capping or length regulation. Consistent with the model that the Stn1p essential function is to promote telomere end protection through Cdc13p, stn1 alleles that truncate the C-terminal 123 residues fail to interact with Cdc13p and do not support viability when expressed at endogenous levels. Remarkably, more extensive deletions that remove an additional 185 C-terminal residues from Stn1p now allow cell growth at endogenous expression levels. The viability of these stn1-t alleles improves with increasing expression level, indicating that increased stn1-t dosage can compensate for the loss of Cdc13p-Stn1p interaction. However, telomere length is misregulated at all expression levels. Thus, an amino-terminal region of Stn1p is sufficient for its essential function, while a central region of Stn1p either negatively regulates the STN1 essential function or destabilizes the mutant Stn1 protein.

Deletion of a trypanosome telomere leads to loss of silencing and progressive loss of terminal DNA in the absence of cell cycle arrest.

Eukaryotic chromosomes are capped with telomeres which allow complete chromosome replication and prevent the ends from being recognized by the repair machinery. The African trypanosome, Trypanosoma brucei, is a protozoan parasite where antigenic variation requires reversible silencing of a repository of telomere-adjacent variant surface glycoprotein (VSG) genes. We have investigated the role of the telomere adjacent to a repressed VSG. In cells lacking telomerase, the rate of telomere-repeat loss appeared to be inversely proportional to telomere length. We therefore constructed strains in which a single telomere could be immediately removed by conditional I-SceI meganuclease cleavage. Following telomere deletion, cells maintain and segregate the damaged chromosome without repairing it. These cells continue to proliferate at the normal rate but progressively lose terminal DNA at the broken end. Although sirtuin-dependent repression is lost along with the telomere, VSG-silencing is preserved. The results provide direct evidence for telomere-dependent repression but suggest a telomere-independent mode of VSG-silencing. They also indicate the absence of a telomere-loss checkpoint in T. brucei.

Cell-cycle-regulated expression and subcellular localization of the Caulobacter crescentus SMC chromosome structural protein.

Structural maintenance of chromosomes proteins (SMCs) bind to DNA and function to ensure proper chromosome organization in both eukaryotes and bacteria. Caulobacter crescentus possesses a single SMC homolog that plays a role in organizing and segregating daughter chromosomes. Approximately 1,500 to 2,000 SMC molecules are present per cell during active growth, corresponding to one SMC complex per 6,000 to 8,000 bp of chromosomal DNA. Although transcription from the smc promoter is induced during early S phase, a cell cycle transcription pattern previously observed with multiple DNA replication and repair genes, the SMC protein is present throughout the entire cell cycle. Examination of the intracellular location of SMC showed that in swarmer cells, which do not replicate DNA, the protein forms two or three foci. Stalked cells, which are actively engaged in DNA replication, have three or four SMC foci per cell. The SMC foci appear randomly distributed in the cell. Many predivisional cells have bright polar SMC foci, which are lost upon cell division. Thus, chromosome compaction likely involves dynamic aggregates of SMC bound to DNA. The aggregation pattern changes as a function of the cell cycle both during and upon completion of chromosome replication.

Recognition and Stabilization of Quadruplex DNA by a Potent New Telomerase Inhibitor: NMR Studies of the 2:1 Complex of a Pentacyclic Methylacridinium Cation with d(TTAGGGT)(4) We thank the EPSRC of the UK and AstraZeneca for financial support to E.G. M.F.G.S. and R.A.H. are supported by the Cancer Research Campaign of the UK.

Telomerase is a cellular ribonucleoprotein, consisting of a reverse transcriptase and an endogenous RNA template, that maintains telomere length at the ends of eukaryotic chromosomes.[1] Telomeres consist of double-stranded DNA composed of tandem repeats of guanine-rich sequences (5 TTAGGG in humans) with a single-stranded 3 -end overhang necessary to ensure complete chromosomal DNA replication, while protecting the chromosome ends from fusion and degradation.[1, 2] The telomeres shorten with each round of cell division, due to incomplete lagging-strand replication, limiting the number of replicative cycles before the cell enters a state of senescence.[3] Telomere length in immortalized cells (e.g. cancer cells) is tightly regulated by the enzyme telomerase whose reverse transcriptase activity allows the addition of 5 -TTAGGG repeats to maintain telomere length through an indefinite number of cell divisions.[4] While telomerase activity is absent in human somatic cells it is detected in the majority of human tumor-derived cell lines. Consequently, telomerase has become a high-profile target for anticancer drug design.[5] The discovery that the G-rich telomeric repeats are able to assemble into novel four-stranded quadruplex structures, consisting of guanine tetrads stabilized by monovalent cations (Na and K ) (Figure 1),[6] has focused attention for structurespecific drug design. Since telomerase reverse transcriptase activity depends on a single-stranded DNA primer,[4] small molecules that bind and stabilize the folded quadruplex form of the primer are potential inhibitors of telomerase function. A number of quadruplex-specific ligands have been reported with the common feature of an extended aromatic ring system capable of interacting through extensive stacking with G-tetrads.[5] However, there has been a paucity of detailed structural data available on the drug ±DNA complexes due to intractable NMR spectra arising from extensive drug-induced line broadening.[7] The exception is a dicationic perylenetetracarboxylic diimide derivative (PIPER), which forms either a sandwich complex bound between the blunt ends of a quadruplex dimer formed from d(TTAGGG)4, or N

Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity

We used a flow cytometric assay to determine the frequency of replication fork arrests during a round of chromosome replication in Escherichia coli. After synchronized initiation from oriC in a dnaC(Ts) strain, non-permissive conditions were imposed, such that active DnaC was not available during elongation. Under these conditions, about 18% of the cells failed to complete chromosome replication. The sites of replication arrests were random and occurred on either arm of the bidirectionally replicating chromosome, as stalled forks accumulated at the terminus from both directions. The forks at the terminal Ter sites disappeared in the absence of Tus protein, as the active forks could then pass through the terminus to reach the arrest site, and the unfinished rounds of replication would be completed without DnaC. In a dnaC2(Ts)rep double mutant, almost all cells failed to complete chromosome replication in the absence of DnaC activity. As inactivation of Rep helicase (the rep gene product) has been shown to cause frequent replication arrests inducing double-strand breaks (DSBs) in a replicating chromosome, DnaC activity appears to be essential for replication restart from DSBs during elongation.

Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations.

Chromosomal rearrangements are found in virtually all types of human cancers. We show that certain chromosome translocations display a delay in mitotic chromosome condensation that is associated with a delay in the mitosis-specific phosphorylation of histone H3. This delay in mitotic condensation is preceded by a delay in both the initiation as well as the completion of chromosome replication. In addition, chromosomes with this phenotype participate in numerous secondary translocations and rearrangements. Chromosomes with this phenotype were detected in five of seven tumor-derived cell lines and in five of thirteen primary tumor samples. These data suggest that certain chromosomal rearrangements found in tumor cells cause a significant delay in replication timing of the entire chromosome that subsequently results in delayed mitotic chromosome condensation and ultimately in chromosomal instability.

Control of chromosomal DNA replication in the early Xenopus embryo.

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle. Over the last decade an enormous amount has been learned about how this is achieved. Much of the progress has come from genetic analysis in yeast. However, biochemical analysis of cell cycle events using extracts prepared from eggs of the South African clawed toad Xenopus laevis has also played an important role. Although there are differences in the detailed regulation of the yeast and frog cell cycles, the basic cell cycle machinery appears to have been conserved throughout evolution. The results obtained in these model organisms, therefore, seem likely to be generally applicable to the cell cycles of all eukaryotes. ### Xenopus eggs and egg extracts The fertilized Xenopus egg undergoes 12 synchronous rounds of cell division in ∼8 h. These cell divisions take place in the absence of growth, subdividing the large (∼1 mm diameter) single‐celled egg into ∼4000 smaller cells. Transcription does not occur during these early embryonic divisions, although translation of pre‐existing maternal mRNA continues. Most of the proteins required for cell cycle progression are pre‐formed in the egg, and the continuing translation of a single protein (cyclin B) can support passage through the whole cell cycle (Murray and Kirschner, 1989). The stockpile of cell cycle proteins present in the Xenopus egg became exploitable by biochemical means following the development of cell‐free extracts that support all the nuclear events of the early embryonic cell division cycle (Lohka and Masui, 1983). Gentle lysis associated with minimal dilution of the cytoplasm yields a ‘low speed supernatant’ that maintains all the cell cycle activities present in the intact egg. These ‘low speed supernatants’ support precise rounds of DNA replication on exogenously added DNA templates, and like the intact egg, only re‐replicate DNA after passage through …

Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times.

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [(3)H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5-15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5-10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.

Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway.

RAD53 and MEC1 are essential genes required for the transcriptional and cell cycle responses to DNA damage and DNA replication blocks. We have examined the essential function of these genes and found that their lethality but not their checkpoint defects can be suppressed by increased expression of genes encoding ribonucleotide reductase. Analysis of viable null alleles revealed that Mec1 plays a greater role in response to inhibition of DNA synthesis than Rad53. The loss of survival in mec1 and rad53 null or point mutants in response to transient inhibition of DNA synthesis is not a result of inappropriate anaphase entry but primarily to an inability to complete chromosome replication. We propose that this checkpoint pathway plays an important role in the maintenance of DNA synthetic capabilities when DNA replication is stressed.