G2 Phase Cell Cycle Checkpoint

The G2 phase checkpoints monitor the integrity of DNA and the accuracy of DNA replication, and the M phase checkpoints ensure correct chromosomal segregation and alignment.

From: Vitamin D (Third Edition) , 2011

Radiation Biology and Radiation Safety

A.C. Begg , in Comprehensive Biomedical Physics, 2014

7.03.2.2.4 Late G2 checkpoint

The late G2 checkpoint describes a long G2 delay that is observed after irradiation and occurs in cells that have been irradiated in the G1 or S phases. These cells may experience transient G1 and S-phase checkpoints but when they arrive in G2 many hours later they experience a second delay before entry into mitosis. Unlike the early G2 checkpoint, this delay is strongly dose dependent and can last many hours after high doses of radiation. In addition, unlike all the other damage checkpoints, this late G2 checkpoint is independent of ATM ( Figure 2(b) ). Instead, the principal signaling occurs from ATR to CHEK1 to CDC25A/C (Xu et al., 2002). The late G2 checkpoint is thus mechanistically similar to the S and early G2 checkpoints, but probably arises from a different type of DNA damage. Instead of being activated by DSB, it is probably caused by damage that persists after other DNA repair processes have been completed and which is sufficient to activate ATR.

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Cell Cycle

Elisa Ventura , Antonio Giordano , in Reference Module in Life Sciences, 2019

G2-Phase Checkpoint

The G2-phase checkpoint, also known as G2/M-phase checkpoint, has the function of preventing cells with damaged DNA, lasting from the G1 and S phases or generated in G2, from undergoing mitosis. The mechanisms acting during the G2-phase checkpoint converge on the inhibition of the mitotic complex CDK1-cyclin B. Different mechanisms may lead to CDK1-cyclin B inhibition, and mainly rely on the inhibition of cdc25 family members by either degradation, mediated by ATM/ATR and CHK1/CHK2, or sequestration, as a consequence of p38 MAPK signaling pathway activation (Mailand et al., 2002; Bulavin et al., 2001). As for cell cycle arrest in G1, cells may undergo both a transient or a prolonged arrest in G2. Prolonged G2 arrest is mainly mediated by the p53-p21 pathway (Taylor and Stark, 2001).

In addition to the described mechanisms, other checkpoint molecules that constitute the so called spindle checkpoint may be activated during the M-phase. The spindle checkpoint senses defects in the attachment of the chromosomes to the spindle and delays the progression through the M-phase to avoid the unequal segregation of chromosomes (London and Biggins, 2014; May and Hardwick, 2006).

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Cellular Responses to DNA Damage

Jean Y.J. Wang , Se Won Ki , in Encyclopedia of Cancer (Second Edition), 2002

II.B G2 Arrest

p53-dependent G2 arrest is mediated by the transcription upregulation of 14-3-3σ. The 14-3-3σ protein belongs to a family of adaptor proteins that are conserved through evolution. Yeast 14-3-3 proteins play an essential role in the G2 checkpoint response by sequestering the inactive Cdc25 phosphatase in the cytoplasm. The mammalian 14-3-3β protein can bind to and sequester Cdc25C in the cytoplasm. The 14-3-3σ protein binds to and retains the cyclin B–Cdc2 complex (MPF) in the cytoplasm. The cyclin B–cdc2 kinase complex must function in the nucleus to initiate mitosis. Thus, the cytoplasmic retention of cyclin B–Cdc2 can inhibit M-phase entry, contributing to a cell cycle arrest in G2 (Fig. 1). The p53-dependent G2 arrest response is distinct from the hChk1-dependent inhibition of Cdc25C. Again, the transcription upregulation of 14-3-3σ by p53 occurs slower than the phosphorylation of Cdc25C by hChk1 (Fig. 1). Thus, p53-dependent G2 arrest may be a secondary mechanism to maintain the G2 checkpoint.

The induction of p21Cip1 by DNA damage also contributes to the maintenance of G2 arrest. Similar to the arrest in G1, this p21Cip1-mediated G2 arrest again requires RB. The mechanism of RB-dependent G2 arrest is likely due to the repression of gene expression. The promoter of the cdc2 gene contains binding sites for E2F. Therefore, RB can inhibit the expression of cdc2, which will enforce the inhibition of mitosis.

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Role of Cyclin B1 Levels in DNA Damage and DNA Damage-Induced Senescence

Yuji Nakayama , Naoto Yamaguchi , in International Review of Cell and Molecular Biology, 2013

3.4 Polyploidization involves the bleomycin-activated ATM/ATR pathway

As G2-arrest is accomplished by bleomycin-activated ATM/ATR, we investigated the correlation of the ATM/ATR pathway and the bleomycin-induced polyploidization (Nakayama et al., 2009). Treatment of cells with caffeine, an ATM/ATR inhibitor, significantly decreases the number of bleomycin-induced polyploid cells. Surprisingly, treatment with caffeine increases the number of dead cells, and knockdown of ATM and ATR kinases by shRNA treatment significantly reduces bleomycin-induced polyploidization and, in turn, enhances cell death. Moreover, the specific inhibitor of Chk1 and Chk2 kinases, debromohymenialdisine, prevents bleomycin-induced polyploidization and enhances cell death. These results suggest that bleomycin-induced polyploidization is mediated by the ATM/ATR pathway. As inhibitory phosphorylation of Cdk1 is not fully sustained upon treatment with bleomycin, cyclin B1 degradation is involved in suppression of Cdk1 kinase activity after dephosphorylation of inhibitory phosphorylation of Cdk1. Thus, we proposed that during bleomycin-induced polyploidization, suppression of Cdk1 activity is achieved by two successive mechanisms: (i) ATM/ATR-induced inhibitory phosphorylation and (ii) cyclin B1 degradation.

Suppression of Cdk1 activity is involved in polyploidization of trophoblasts and megakaryocytes (Edgar and Orr-Weaver, 2001). In addition to inhibition of mitotic entry, suppression of Cdk1 activity is required for the assembly of prereplication complexes for licensing the DNA for another round of replication (Diffley, 2004). Thus, the inactivation of Cdk1 is responsible for bleomycin-induced polyploidization through sustained inhibition of mitotic entry, and possibly through licensing for DNA replication in bleomycin-treated cells. In some types of cells, such as human megakaryocytes, Drosophila follicle cells, and yeasts, activation of APC-mediated proteolysis is involved in polyploidization (Edgar and Orr-Weaver, 2001), and nonperiodic activation of APC causes polyploidization in human cells as well (Sorensen et al., 2000). As polyploidization involves the degradation of geminin, an APC substrate that inhibits the initiation of DNA replication (McGarry and Kirschner, 1998), activation of protein degradation in response to DNA damage therefore may play a role in the induction of polyploidization.

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Regulation of the Cell Cycle

Yan Li , ... J. Alan Diehl , in The Molecular Basis of Cancer (Fourth Edition), 2015

Targeting Cell Cycle Checkpoints

Targeting the S and G2 checkpoints is also attractive for cancer therapy because loss of G1 checkpoint control is a common feature of cancer cells, 89 making them more reliant on the S and G2 checkpoints to prevent DNA damage–triggered cell death. Various molecules such as CHK1, CHK2, PP2A, Wee1, and cell division cycle 25 (CDC25) have been suggested as the key targets for checkpoint abrogation. 90 Numerous checkpoint inhibitors have entered into clinical trials, most of which focused on CHK1. Among all the checkpoint inhibitors, UCN-01 is most clinically advanced, 91-93 but after phase II trials it was discontinued because of dose-limiting toxicities and a lack of convincing efficacy. The newer, more specific inhibitors of CHK are still under investigation. 90,94

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Inhibition of DNA Repair as a Therapeutic Target

Stephany Veuger , Nicola J. Curtin , in Cancer Drug Design and Discovery (Second Edition), 2014

ATR and CHK1

Targeting the S and G2 checkpoints is particularly attractive for cancer therapy because loss of G1 checkpoint control is a common feature of cancer cells, for example, due to defects in the p53 and pRb tumor suppressor genes or an imbalance in cyclins, cyclin-dependent kinases, and their inhibitors. This makes cancer cells more reliant on their S and G2 checkpoints to prevent DNA damage from being translated into cell death [243–245]. Proof-of-principle genetic studies show that dominant negative inhibition of ATR with a kinase-dead ATR mutant, or employing CHK1 small interfering RNA (siRNA) led to abrogation of DNA damage-induced G2 arrest and sensitization of cells to a variety of DNA-damaging chemotherapeutic agents [246–251]. Sensitization was specific to replicating cells, and in some studies inhibition of the ATR–CHK1 pathway selectively sensitized cells that were defective in the G1 checkpoint.

Despite the attractiveness of the target, small-molecule inhibitors of ATR have proved elusive [252]. This may reflect the difficulty of assaying an enzyme that requires a complex of coactivators and regulators, and the progress of ATR research has been hampered by the lack of potent inhibitors. Caffeine, the prototype inhibitor, was weak and nonspecific but provided data that were sufficiently promising for the target to be pursued [253]. Schisandrin B, a natural product, was identified as inhibiting ATR, abrogating the ultraviolet (UV)-induced S and G2/M checkpoint and increasing UV cytotoxicity in human lung cancer cells [254]. In a screen of PI3K inhibitors, PI103 and PI124 were identified as being potent ATR inhibitors with IC50 values of 0.9 and 2   µM, respectively [255] (Fig. 8.11). In a HTS assay NVP-BEZ235, which was previously thought to be selective for PI3K and mTOR, was demonstrated to be a potent inhibitor of ATR (IC50  =   100   nM); and the most potent ATR inhibitor, ETP-46464 (IC50  =   25   nM), inhibited the restart of stalled replication forks and abrogated S phase arrest after hydroxyurea exposure [256]. VE-821, AZ-20, and NU6027 have recently been identified as being ATR inhibitors [257–260]. All drugs inhibited CHK1 phosphorylation at Ser345 and sensitized cells to a variety of DNA-damaging agents. VE-821 enhanced IR-induced cytotoxicity in a panel of 12 human cancer cell lines, caused a more profound radiosensitization in hypoxic cells. It also increased reoxygenation-induced DNA damage and decreased the survival of cells undergoing reoxygenation [261]. Interestingly, NU6027 inhibited RAD51 focus formation (indicative of HRR suppression) and was more cytotoxic in the presence of a PARP inhibitor or when XRCC1 was defective, suggesting the potential for synthetic lethality. AZ-20 is reported to be an even more potent ATR inhibitor with an IC50 of 4.5   nM in biochemical assays and 51   nM in cellular assays. This inhibitor was active as a single agent both in vitro and in vivo, and, at an oral dose of 25   mg/kg bid or 50   mg/kg qd, it inhibited the growth of LoVo xenografts. This is the first report of an ATR inhibitor in an in vivo model, and although only published in abstract form, the full data on this compound are eagerly awaited.

FIGURE 8.11. ATR and CHK1 inhibitors. Compounds to the left of the vertical line are ATR inhibitors; those to the right are CHK1 inhibitors.

There have been substantial research efforts into the development of CHK1 inhibitors that have culminated in clinical studies. These include the nonspecific staurosporin analog UCN-01 and its derivatives, such as PD321852, the potent dual CHK1 and CHK2 inhibitors AZD7762 and XL9844, and the highly potent selective CHK1 inhibitors PF00477736, CEP-3891, SAR-020106, and SCH900776 (reviewed in Refs [262–265]). When used as a single agent, most of these inhibitors do not affect cell cycle distribution and are not cytotoxic. However, they do prevent cell cycle arrest and increase cytotoxicity after exposure to DNA-damaging agents, including IR and those causing replication stress such as antimetabolites (e.g., gemcitabine), topoisomerase I poisons, and DNA cross-linking agents (e.g., cisplatin), suggesting that the S phase checkpoint is critical. Early studies with UCN-01 demonstrated an abrogation of doxorubicin-induced G2/M arrest and potentiation of cisplatin and camptothecin cytotoxicity in human cancer cell lines [266–268]. The more potent and selective CHK1 inhibitor, SAR-020106 (Sareum) (IC50 of 13.3   nM), blocked etoposide-induced cell cycle arrest in HT29 cells [269]; and PF00477736 abrogated gemcitabine- and camptothecin-induced S phase and G2/M arrest, and potentiated the activity of a variety of DNA-damaging agents in several human cancer cell lines [270]. The dual CHK1 and CHK2 inhibitor, AZD7762, inhibited camptothecin-induced G2 arrest and potentiated the cytotoxicity of gemcitabine and topotecan [271,272]. XL-844 blocked gemcitabine-induced S-phase arrest, resulting in premature entry into mitosis [273]. CHK1 inhibitors are also radiosensitizers: AZD7762 markedly increased IR cytotoxicity in a panel of prostate, lung, and colon cancer cell lines [274]; CEP-3891 prevented IR-induced S and G2 arrest in U2OS cells, and increased nuclear fragmentation and cytotoxicity after IR [275].

These in vitro studies translated into positive in vivo xenograft studies, mostly in combination with gemcitabine or IR. For example, PF-00477736 increased the efficacy of gemcitabine against CoLo205 xenografts [270], AZD7762 increased the antitumor activity of gemcitabine and of irinotecan against human cancer xenografts in both mice and rats [272], and XL-844 increased the efficacy of gemcitabine against PANC-1 xenografts [273]. SCH900776 increased gemcitabine-induced DSB accumulation and enhanced the anticancer activity of gemcitabine without exacerbating gemcitabine-induced myelosuppression [276]. Interestingly, SAR-020106 potentiated the antitumor activity of gemcitabine against SW620 xenografts better if administered at the same time than if delayed by 24   h. In vivo radiosensitization data are scarce, but AZD7762 demonstrated radiosensitizing activity in xenograft models of lung cancer with brain metastasis resulting in prolonged survival [269,277].

UCN-01 was the first CHK1 inhibitor to undergo clinical evaluation as a single agent; short infusions were better tolerated than long ones, but after some phase II trials as a single agent and in combination studies, this agent has been discontinued [277,278]. Several of the second-generation inhibitors have been in phase I trials, mostly in combination with gemcitabine, but largely have been reported only in abstract form. Hematological toxicity (neutropenia and thrombocytopenia) was commonly seen with PF00477736 and SCH900776 in combination with gemcitabine and with AZD7762 in combination with gemcitabine and irinotecan (reviewed in Ref. [279]).

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Protein-Protein Interactions in Human Disease, Part B

Iva Ugrinova , ... Philippe Bouvet , in Advances in Protein Chemistry and Structural Biology, 2018

5 Functions of Nucleolin in Cell Division

Nucleolin depletion leads to cell cycle arrest (G2/M in HeLa and G1 in DT40 cells) (Ma et al., 2007; Storck, et al., 2009; Ugrinova et al., 2007). In addition to numerous nuclear alterations, including the presence of micronuclei, multiple nuclei, or large nuclei, a defect in the control of centrosome duplication is observed (Ugrinova et al., 2007). Nearly a century ago, Boveri (1914) proposed that tumors develop as a consequence of chromosomal imbalances, suggesting that centrosome aberrations could be the cause of such imbalances (Boveri, 1914). Nowadays, many human tumors have been described to carry extensive centrosome aberrations, and a strong correlation between the extent of these aberrations and the clinical aggressiveness of tumors has been reported (Lingle, Lukasiewicz, & Salisbury, 2005; Nigg, 2002; Zyss & Gergely, 2009). Bipolar mitotic spindle is important for proper chromatid separation during mitosis. It is worth mentioning that many cells use centrosome-independent spindle assembly mechanisms to cluster extra centrosomes and to allow the formation of bipolar spindles (Gadde & Heald, 2004; Kwon et al., 2008; Quintyne, Reing, Hoffelder, Gollin, & Saunders, 2005; Ring, Hubble, & Kirschner, 1982). Nevertheless, numerical centriole aberrations and centrosome aneuploidy are important sources of chromosomal instability in tumor cells (Nigg, Cajanek, & Arquint, 2014; Pihan, 2013). It has been reported that nucleolin is involved in centrosome misregulation (Gaume et al., 2015; Ugrinova et al., 2007). The knocking down of nucleolin by RNA interference leads to abnormal centrosome amplification and to the formation of multipolar mitotic spindles (Ma et al., 2007; Ugrinova et al., 2007). Several groups have reported experiments suggesting the implication of nucleolin in centrosome regulation. Nucleolin has been associated to human centrosomes (Andersen et al., 2003) and mitotic spindles (Sauer et al., 2005) in different proteomic studies. By immunofluorescence, a phosphorylated form of nucleolin has been detected in mitosis at the spindle poles (Ma et al., 2007). Interestingly, in interphase, nucleolin was found to colocalize only with the mature centrioles and its depletion to induce an amplification of immature centriole markers that were therefore unable to nucleate microtubules (Gaume et al., 2015). Furthermore, nucleolin was coimmunoprecipitated with centrosomal proteins—γ-tubulin and ninein, a protein of the subdistal appendages involved in microtubule anchoring (Gaume et al., 2015). In addition, during microtubule growth phases, nucleolin affects microtubule stabilization by modifying both the speed and lifetime of polymerization and by reducing catastrophe frequency (Gaume et al., 2016).

Altogether, these data suggest that nucleolin is involved in microtubules anchoring to centrosomes in interphase cells and microtubules polymerization (Gaume et al., 2016, 2015). Although the centrosome misregulation is associated with tumorigenicity, the potential effects of centrosomal nucleolin on cancer cell apparition or progression have never been addressed (Berger, Gaume, & Bouvet, 2015).

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Adult and Fetal

Francis W. Ruscetti , Stephen H. Bartelmez , in Handbook of Stem Cells, 2004

HEMATOPOIETIC CYTOKINES AND "OVERRIDING THE CHECKPOINT"

The arrest at the G1 and G2 checkpoint responses is thought to occur to allow DNA damaged cells time to either repair the damage, particularly to DNA, or alternatively undergo apoptosis or senescence. Support for this hypothesis comes from the ability of hematopoietic cytokines, such as IL-3, SCF, and erythropoietin (EPO), to prevent G1 and G2 checkpoint delays after exposures to IR and chemotherapeutic agents. 139 Treatment of bone marrow cells with both IL-3 and DNA-damaging agents prevents cells from undergoing apoptosis. In hematopoietic cell lines, apoptosis and G2/M arrest induced by IR can be rescued by cytokines through distinct JAK kinase signaling pathways. 140 In contrast, DNA damage induced G1 arrest in these same cells is alleviated by cytokine-mediated PI3K-dependent activation of Cdk2. 141 It would be predicted that the lack of checkpoint delay might greatly increase cell death. Continuous cytokine activation of PI3K in the presence of IR and cisplatin DNA damage results in greatly enhanced lethality of the DNA-damaging agent in hematopoietic cells. 142, 143 Furthermore, in the autosomal recessive disease AT, the loss of ATM function results in attenuation of the G1, S, and G2 checkpoint functions in response to IR. 144 Cells from individuals with AT are hypersensitive to the killing effects of IR and display 3- to 10-fold increased frequencies in genetic instability. 145

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Histone Deacetylase Inhibitors as Cancer Therapeutics

Brian Gabrielli , Mellissa Brown , in Advances in Cancer Research, 2012

2.4 HDACi-sensitive G2 phase arrest

In cell lines that are competent for the HDACi-induced G2 phase arrest, the arrest is triggered by the continuous presence of HDACi throughout S phase (Lallemand et al., 1999; Qiu et al., 2000; Richon et al., 2000). The arrest operates independently of the well-studied DNA damage-induced G2 checkpoint arrest mechanisms, as DNA damaging agents can initiate a G2 phase checkpoint arrest in cells that failed to arrest in response to HDACi treatment (Qiu et al., 2000). The mechanism by which the HDACi impose the G2 phase arrest is unclear. A range of different mechanisms have been demonstrated to trigger a G2 phase arrest including: downregulation of G2/M phase Cyclins (Maity et al., 1996), increased expression of Cdk inhibitor proteins such as p21 (Bunz et al., 1998), and activation of checkpoint signaling through either ATM-Chk2, ATR-Chk1 (Niida & Nakanishi, 2006) or p38MAPK–MK2 (Manke et al., 2005). Expression of Epstein–Barr virus latent proteins disables ATM/ATR-dependent checkpoint signaling and the HDACi-induced G2 phase arrest (Krauer et al., 2004), which suggests the involvement of the canonical ATM/ATR checkpoint signaling pathway. However, there is no evidence that either ATM/ATR or downstream Chk1 or Chk2 signaling is activated by HDACi treatment (Lee, Choy, Ngo, Venta-Perez, & Marks, 2011; Mikhailov, Shinohara, & Rieder, 2004).

HDACi treatment has been shown to block the activation of the G2/M complexes, Cyclin A/Cdk2 and Cyclin B/Cdk1, and decrease Cyclin B1 protein levels (Lallemand et al., 1999; Qiu et al., 2000). Acetylation of Cyclin A reportedly destabilizes the protein (Mateo et al., 2009), which is likely to be responsible for reducing the levels of this critical G2 phase regulator. The protein level of Aurora A has also been reported to be reduced with HDACi treatment, possibly as a consequence of HSP90 inhibition by decreased association with HDAC6 (Cha et al., 2009). Similarly, the expression of Plk1 itself has also been shown to be down regulated in response to HDACi treatment (Lallemand et al., 1999; Prystowsky et al., 2009; Fig. 1.3). Finally, the expression of Gadd45, a growth arrest and DNA damage-inducible gene that can cause a G2/M arrest by inhibiting cdc2/Cyclin B activity (Jin et al., 2000), has also been reported to be upregulated following HDACi treatment, and may also participate in the G2 arrest (Hirose et al., 2003).

Figure 1.3. With HDACi treatment, the levels of the indicated mitotic regulators are reduced, either through reduced mRNA levels or protein stability, producing a G2 phase arrest in the subset of cell lines capable of triggering a G2 phase arrest in response to HDACi. The molecular basis determining the ability to impose this arrest is unclear, but is present in normal cell types.

A second G2 checkpoint mechanism is induced upon HDACi treatment when cells are in antephase, which occurs immediately prior to nuclear membrane breakdown. This arrest utilizes the p38MAPK–MAPKAPK2 pathway to inhibit entry into mitosis (Manke et al., 2005; Mikhailov et al., 2004). This mechanism, the antephase checkpoint, appears to be distinct from the G2 phase checkpoint initiated in response to S phase HDACi addition.

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Organization, Synthesis, and Repair of DNA

John W. Pelley PhD , in Elsevier's Integrated Biochemistry, 2007

Formation of the Replication Fork

Entry into the S phase initiates the process of DNA replication. Since the two strands of the DNA helix must separate to serve as templates (semiconservative replication), the higher order packing of the chromatin must be reduced to allow access by the replication enzymes. Semiconservative replication results in one parent (original) strand and one daughter (new) strand in each new double helix.

HISTOLOGY

Mitosis (M)

After a cell has passed the G2 checkpoint, it must also pass an M checkpoint that detects improper spindle formation in order to prevent mis-segregation of the chromatids to daughter cells. If the M checkpoint is passed, cells may enter mitosis and proceed through metaphase (where chromosomes line up on metaphase plate) and anaphase (where chromosomes separate as they are pulled to opposite spindle poles).

HISTOLOGY

Apoptosis

Programmed cell death, apoptosis, refers to an orderly, natural process by which cells commit suicide. For example, after only one day of existence, neutrophils form blebs on their surface that are digested by other phagocytic cells. Their DNA also undergoes degradation and digestion. Another event seen during the stepwise process of cellular apoptosis is mitochondrial degradation.

Since the two strands of the DNA helix are antiparallel, each direction contains a template strand. Thus, DNA synthesis is bidirectional starting from an origin of replication for both eukaryotic and prokaryotic DNA (Fig. 15-3). Eukaryotic DNA synthesis differs primarily by having multiple origins of replication in order to reduce the time necessary to replicate the much larger chromosome.

For the DNA helix to be accessible by polymerization enzymes, the supercoiling is relaxed by the action of DNA gyrase (topoisomerase II), an enzyme that induces negative (opposite direction to right-handed twist) supercoils in DNA. The relaxation of the supercoiled DNA allows helicase to bind and continue to unwind the helix in an energy-requiring reaction (Fig. 15-4). Helicase is not a topoisomerase, since it does not break and rejoin the strands—it simply spreads the DNA strands apart. This action results in the formation of positive supercoils (same direction as right-handed twist) ahead of the replication fork. This can be illustrated by quickly pulling apart two strands from a string or rope, causing a knot (the positive supercoil) to form ahead of the separation (Fig. 15-5). Topoisomerase I relieves the strain by repeatedly breaking and rejoining one strand of the helix as it unwinds one turn, and thus is capable of relaxing either positive or negative supercoils. As helicase unwinds the helix, helix-destabilizing proteins bind to the single-stranded DNA to prevent reannealing, and the point of separation is referred to as a replication fork.

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