By:Sapna Das-Bradoo, Ph.D.&Anja-Katrin Bielinsky, Ph.D.(Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota)© Education
Citation:Das-Bradoo,S.&Bielinsky,A.(2010)DNA Replication and Checkpoint Control in S Education3(9):50





During DNA replication, the unwinding of strands leaves a single strand vulnerable. How does the cell protect these strands from damage?

Replicating DNA is fragile, and can break duringthe duplication process. In fact, broken chromosomes are often the source ofDNA rearrangements and can change the genetic program of a cell. These changescan trigger a growth advantage in a single cell in your body, and when thatcell continues to divide, tumors arise. Fortunately, our cells have defensemechanisms to shield us from these damaging events.

In theeukaryotic cell cycle, chromosome duplication occurs during "S phase" (thephase of DNA synthesis) and chromosome segregation occurs during "Mphase" (the mitosis phase). During S phase, any problems with DNAreplication trigger a ‘"checkpoint" — a cascade of signaling events that puts thephase on hold until the problem is resolved. The S phase checkpoint operateslike a surveillance camera; we will explore how this camera works on themolecular level. The last 60 years of research in bacterial species(specifically, Escherichia coli) andfungal species (specifically, Saccharomycescerevisiae), have continually demonstrated that several major processesduring DNA replication are evolutionarily conserved from bacteria to highereukaryotes.

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Before delving into the intricacies ofcheckpoints, we must remind ourselves of the key molecules and processes of DNAreplication. What happens to DNA when it is duplicated?

Recall thatchromosomes are made of double-stranded (ds) DNA. How does thecell duplicate two strands of identical DNA copies simultaneously? The goal ofreplication is to produce a second and identical double strand. Because each ofthe two strands in the dsDNA molecule serves as a template for a new DNA strand,the first step in DNA replication is to separate the dsDNA. This isaccomplished by a DNA helicase. Once the DNA template is single-stranded (ss),a DNA polymerase reads the template and incorporates the correctnucleoside-triphosphate in the opposite position (Figure 1). Because of thecharacteristic y-shape of the replicating DNA, it is often referred to as a"replication fork." Particularly important are two aspects of the replicationfork: 1) the 5" to 3" polarity of the newly synthesized DNA and 2) the sequenceof base pairs (color-coded in Figure 1). The DNA code in each of the strands isthe same, but inverted, so that the sequence is identical when read in the 5"to 3" direction. This is the direction in which all DNA is polymerized, andalso the direction in which a DNA sequence is read when written out, byconvention.
(A) Nucleoside triphosphates serve as a substrate for DNA polymerase, according to the mechanism shown on the top strand. Each nucleoside triphosphate is made up of three phosphates (represented here by yellow spheres), a deoxyribose sugar (beige rectangle) and one of four bases (differently colored cylinders). The three phosphates are joined to each other by high-energy bonds, and the cleavage of these bonds during the polymerization reaction releases the free energy needed to drive the incorporation of each nucleotide into the growing DNA chain. The reaction shown on the bottom strand, which would cause DNA chain growth in the 3" to 5" chemical direction, does not occur in (B) DNA polymerases catalyse chain growth only in the 5" to 3" chemical direction, but both new daughter strands grow at the fork, so a dilemma of the 1960s was how the bottom strand in this diagram was synthesized. The asymmetric of the replication fork was recognized by the early 1970s: the leading strand grows continuously, whereas the lagging strand is synthesized by a DNA polymerase through the backstitching mechanism illustrated. Thus, both strands are produced by DNA synthesis in the 5" to 3" direction.
© 2002 From Molecular Biology of the Cell, 4th Edition by Alberts et al. Reproduced with permission of Garland Science/Taylor & Francis LLC. All rights reserved.

The DNA strandthat is synthesized in the 5" to 3" direction is called the leading strand. Theopposite strand is the lagging stand, and although it is also synthesized inthe 5" to 3" direction, it is assembled differently. As a rule, none of theknown DNA polymerases adds a nucleoside triphosphate onto a free 5" end. This brings us to the first rule of DNAreplication: DNA synthesis only occursin one direction, from the 5" to the 3" end.

Applying thisrule helps us understand why the lagging strand is generated from a series ofsmaller fragments (Figure 1b). These fragments are known as Okazaki fragments, after Reiji and TsunekoOkazaki, who first discovered them in 1968. Each time the DNA fork opens, the leadingstrand can be elongated, and a new Okazakifragment is added to the lagging strand.All Okazakifragments are subsequently joined together by DNA ligase to form a longcontinuous DNA strand (Anderson & DePamphilis 1979; Alberts 2003). In thisregard, eukaryotic DNA replication follows the same principles as prokaryoticDNA replication.

Amongst the arrayof proteins at the replication fork, DNA polymerases are central to the processof replication. These important enzymes can only add new nucleosidetriphosphates onto an existing piece of DNA or RNA; they cannot synthesize DNA de novo (from scratch), for a giventemplate. Another class of proteins fills this functional gap. Unlike DNApolymerases, RNA polymerases can synthesize RNA de novo, as long as a DNA template is available. This particularfeature of de novo synthesis issimilar to what happens during mRNA transcription.

Eukaryoticcells possess an enzyme complex that has RNA polymerase activity, but works inDNA replication. This unique enzyme complex is called DNA primase.Interestingly, this primase generates small 10-nucleotide-long RNA primers froma DNA template (the red portion of the Okazakifragment in Figure 2). The RNA primers produced are later replaced by DNA, sothat the newly-synthesized lagging strand is not a mixture of DNA and RNA, butconsists exclusively of DNA. The chemical properties of DNA and RNA are quitedifferent, and DNA is the preferred storage material for the geneticinformation of all cellular organisms, so this reinstallment of a continuousDNA strand is very important.

In prokaryoticcells, DNA primase is its own entity and works in a complex with the DNAhelicase (Figure 2) (Alberts 2003; Langston & O"Donnell 2006). However, ineukaryotic cells DNA primase is associated with another polymerase, DNApolymerase-α | | | pol-α | | |, which initiates the leading strand and all Okazaki fragments (Pizzagalli, al. 1988; Hubscher, Maga, &Spadari 2002).At present, we have no evidence that DNA primase binds to the DNA helicase ineukaryotic cells. But it is likely that some connector protein coordinates DNAunwinding and DNA synthesis initiation in eukaryotic cells.
These proteins are illustrated schematically in panel a of the figure below, but in reality, the fork is folded in three dimensions, producing a structure resembling that of the diagram in the inset b. Focusing on the schematic illustration in a, two DNA polymerase molecules are active at the fork at any one time. One moves continuously to produce the new daughter DNA molecule on the leading strand, whereas the other produces a long series of short Okazaki DNA fragments on the lagging strand. Both polymerases are anchored to their template by polymerase accessory proteins, in the form of a sliding clamp and a clamp loader. A DNA helicase, powered by ATP hydrolysis, propels itself rapidly along one of the template DNA strands (here the lagging strand), forcing open the DNA helix ahead of the replication fork. The helicase exposes the bases of the DNA helix for the leading-strand polymerase to copy. DNA topoisomerase enzymes facilitate DNA helix unwinding. In addition to the template, DNA polymerases need a pre-existing DNA or RNA chain end (a primer) onto which to add each nucleotide. For this reason, the lagging strand polymerase requires the action of a DNA primase enzyme before it can start each Okazaki fragment. The primase produces a very short RNA molecule (an RNA primer) at the 58 end of each Okazaki fragment onto which the DNA polymerase adds nucleotides. Finally, the single-stranded regions of DNA at the fork are covered by multiple copies of a single-strand DNA-binding protein, which hold the DNA template strands open with their bases exposed. In the folded fork structure shown in the inset, the lagging-strand DNA polymerase remains tied to the leading-strand DNA polymerase. This allows the lagging-strand polymerase to remain at the fork after it finishes the synthesis of each Okazaki fragment. As a result, this polymerase can be used over and over again to synthesize the large number of Okazaki fragments that are needed to produce a new DNA chain on the lagging strand. In addition to the above group of core proteins, other proteins (not shown) are needed for DNA replication. These include a set of initiator proteins to begin each new replication fork at a replication origin, an RNAseH enzyme to remove the RNA primers from the Okazaki fragments, and a DNA ligase to seal the adjacent Okazaki fragments together to form a continuous DNA strand.
© 2002 From Molecular Biology of the Cell, 4th Edition by Alberts et al. Reproduced with permission of Garland Science/Taylor & Francis LLC. All rights reserved.

After strandinitiation, other DNA polymerases continue DNA elongation. In eukaryotic cells,these polymerases cooperate with a sliding clamp called proliferating cellnuclear antigen (PCNA). The regulation of PCNA is highly complexand important for DNA replication and repair (Moldovan, Pfander, & Jentsch2007).There may be additional, yet undiscovered, parallel (or identical) mechanismsor proteins that coordinate DNA unwinding and DNA elongation. Observations insimpler model organisms strongly hint that eukaryotes too have a connectingmechanism that coordinates DNA helicase, and a DNA polymerase-a/DNA primase (pol-a/primase)complex.

How would youidentify the protein that serves as a connector between DNA helicase and pol-a/primase? A simple yet often effective approach is to findproteins that directly bind to both enzymes. However, that requires us tounderstand the molecular architecture of DNA helicase.

In eukaryotes,the DNA helicase is comprised of a structural core and two regulatory subunits.The core, which contains the ATP hydrolysis activity, is a hexameric complexformed of the minichromosome maintenance proteins 2-7,called Mcm2-7 (Bochman& Schwacha 2008; Bochman & Schwacha 2009; Schwacha & Bell 2001). Mcm2-7encircles dsDNA (Remus et al.2009),but remains inactive until two additional regulatory subunits assemble onto it.Those factors are cell division cycle protein 45 (Cdc45) and GINS (Go,Ichi, Ni, and San; Japanese for "five, one, two, and three," which refers tothe annotation of the genes that encode the complex). Scientistscall this resulting functional DNA helicase a CMG complex (formed by Cdc45,Mcm2-7, GINS) (Moyer,Lewis, & Botchan 2006). Inprinciple, any of these assembled components could be linked to pol-a/primase by a hypothetical connector protein. Scientistshave actually identified two candidate connector proteins that directly bind toboth helicase and primase: 1) Mcm10 (another Mcm protein that, despite its name,has no functional resemblance to any of the Mcm2-7 proteins) (Solomon et al. 1992.; Merchant et al. 1997) and 2) chromosometransmission fidelity protein 4 (Ctf4) (Kouprina et al. 1992).Specifically, both of these proteins interact with pol-a/primase (Fien et al. 2004;Ricke & Bielinsky 2004; Warren etal. 2009; Miles & Formosa 1992) and CMG complex subunits (Merchant et al. 1997; Gambus et al. 2009). In budding yeast, Mcm10 is essential for replication tooccur. However, in these same cells DNA replication can function normallywithout Ctf4, which means that Ctf4 is not absolutely required (Kouprina et al. 1992). What abouthigher eukaryotes? Other experiments in human cells have shown that bothproteins seem to be necessary, and work together during replication (Zhu, et al. 2007). Scientistsare still actively investigating these complex mechanisms.

Why iscoordination between DNA unwinding and synthesis important? What would happenif you lose this coordination? Because pol-a/primasealways requires CMG function to create the ssDNA template, it could neversurpass the DNA helicase (Figure 2b). Without a connecting link, the CMGcomplex could just "run off" and leave pol-a/primasebehind. This would create long regions of vulnerable ssDNA. Therefore, thesecond rule in DNA replication is that DNAunwinding and DNA synthesis have to be coordinated.

Figure 3:Single-stranded DNA (ssDNA) gaps with a 5" primer end are formed during nucleic acid metabolism
© 2008 Publishing Group Cimprich, K. A. & Cortez, D. ATR: an essential regulator of genome integrity. Reviews Molecular Cell Biology 9, 616–627 (2008) doi:10.1038/nrm2450. All rights reserved.
As mentionedabove, a checkpoint is a cascade of signaling events that puts replication onhold until a problem is resolved. How does a cell know that there is a problemwith replication? dsDNA is intrinsically more stable than ssDNA, although thelatter can be stabilized and protected by single-strand DNA binding proteins.Researchers have recently discovered that, in eukaryotes, the replicationprotein A (RPA) is a form of red flag in the cell: when RPA is coating longstrands of ssDNA, this signals a checkpoint. This concept underscores animportant feature: presence of ssDNAsignals that "something is wrong" and this also holds true for other phases ofthe cell cycle. In other words,whether ssDNA is created during replication, or outside of S phase, it willalways trigger the checkpoint surveillance system (Figure 3). Interestingly, this phenomenon is also presentat unprotected telomeres (chromosomeends) that contain ssDNA (Figure 3).

What is themechanism of a red flag, or danger signal that activates a checkpoint? How doesit alert the cell? Scientists who have asked this question don"t know the entireanswer, but they have learned that RPA-coated ssDNA attracts a specific proteinwith a complicated name: the ataxia telangiectasia mutated and Rad3related kinase, also known as ATR (Cimprich & Cortez 2008). ATRassociates with RPA and activates its intrinsic kinase activity. This starts a thattemporarily halts S phase progression. Therefore, ATR is also known as the Sphase "checkpoint kinase."

ATR kinaseacts in several ways to keep the replication process intact. There is evidencethat ATR also stabilizes replication forks that contain ssDNA (Katou et al. 2003). How thishappens remains largely unclear, but recent evidence suggests that ATR mayaffect the Mcm2-7 proteins, the inner core of the CMG helicase mentioned above(Cortez,Glick, & Elledge 2004; Yoo et al.2004).One hypothesis is that phosphorylation of one or several of the Mcm2-7 subunitsprevents the CMG complex from unwinding more and more DNA. This actioneffectively stops the process so that it can be repaired before proceeding.Currently, many researchers are trying to better understand the mechanisms ofcrosstalk between ATR and the replication machinery (Forsburg2008; Bailis et al. 2008).

Figure 4:Stalled replication forks activate the ataxia-telangiectasia mutated and RAD3-related (ATR) kinase
Nucleases can cleave stalled forks, causing double-strand breaks (DSBs) to form and activate ataxia-telangiectasia mutated (ATM). The rate at which DSBs form at stalled forks is greatly increased in cells with defective ATR signalling.
© 2008 Publishing Group Cimprich, K. A. & Cortez, D. ATR: an essential regulator of genome integrity. Reviews Molecular Cell Biology 9, 616-627 (2008) doi:10.1038/nrm2450. All rights reserved.
In normalcells, the uncoupling of DNA unwinding and DNA polymerization resulting inssDNA is actually a rare event. So why would normal cells need ATR? There areother circumstances that cause replication to go awry. One is that the DNAtemplate somehow becomes defective during replication, and causes thepolymerase to pause (Figures 3 and 4a). For example, a DNA base can bechemically modified or spontaneously altered. This generates a lesion — an areathat is a roadblock for DNA polymerases and DNA primase. Therefore, DNA lesionscause regions of DNA to remain single-stranded (uncopied).

Scientists usethe term "stalled forks" for areas of replication forks where DNApolymerization is halted. Stalled forks activate ATR, which in turnphospohorylates its downstream target, the checkpoint kinase 1 (Chk1) (Figure4) (Cimprich& Cortez 2008). Little is known about the phosphorylation targets that liefurther downstream of Chk1, but when scientists observe Chk1 phosphorylation incells, they conclude that cells are actively trying to protect replicationforks with DNA lesions.

What happenswhen ATR function goes awry? Normally, once DNA polymerization resumes andssDNA is converted into dsDNA, ATR is inactivated and cells are released fromthe checkpoint. However, if the ATR signaling pathway is defective, due to amutation in ATR or Chk1 (Menoyo et al.2001),then ssDNA is converted into a double-strand break (DSB), a complete cleavageof both DNA strands (Figure 4, right).

A DSB is acatastrophic event because it ruins the replication fork. Under thesecircumstances, cells activate the ATM kinase (Figure 4, on the right). Asmentioned above, ATM and ATR are related to each other as they share some aminoacid sequences (Shiloh 2003), but ATM has a different function: itworks exclusively to repair DSBs (Cimprich & Cortez 2008). It does soby phosphorylating checkpoint kinase 2 (Chk2), a protein that triggers acascade of phosphorylation events that ultimately result in the repair of theDSB. Only if the DSB is successfully repaired can DNA replication resume.

Interestingly,when Chk2 triggers events that ultimately repair a DSB, another event alsotakes place. This event is the phosphorylation of the well-known p53 (Caspari 2000). Thisobservation is a clue that repairing DSBs may have something to do withpreventing the formation of tumors.

Together with a variety of othermolecules, ATR and ATM kinases are key factors for the surveillance of DNAreplication, and prevent chromosome breakage in dividing cells. However, duringrepair processes, chromosome fragments can be improperly joined together.Indeed, some scientists consider that such mistakes enable some degree ofgenetic evolution by creating new and different genetic sequences.Nevertheless, if even a single cell in our body makes a mistake and fuses DNAfragments to each other that are not supposed to be joined, the rearrangementcan be sufficient to deregulate normal cell division. If multiple changes ofthis type accumulate, then this single cell can eventually turn into atumor.

Given thisunderstanding, would it be true that people who carry a mutation in the ATM,ATR, CHK1, or CHK2 genes have a higher risk of developing cancer? Yes. In theseaffected individuals, the cellular surveillance system described above isdefective and no longer provides full protection from random events that affectDNA replication. For example, the name of the ATM protein derives from the afflictionthat results from a mutated ATM protein: ataxia telangiectasia. In thisdisease, patients suffer from motor and neurological problems, and they alsohave what is known as a genome instability syndrome that geneticallypredisposes them to developing cancer (Shiloh 2003). In addition,when scientists examine cells directly, the experimental inhibition of ATM,ATR, Chk1, Chk2, or the connector protein Mcm10 causes a very dramatic increaseof DSBs (Paulsen et al. 2009; Chattopadhyay &Bielinsky 2007). With these observations, it may be possible to create newideas for novel diagnostics and therapies for cancer that specifically trackthese potent molecules.

The process ofDNA replication is highly conserved throughout evolution. Investigating thereplication machinery in simple organisms has helped tremendously to understandhow the process works in human cells. Major replication features in simplerorganisms extend uniformly to eukaryotic organisms, and replication followsfundamental rules. During replication, complex interactions between signalingand repair proteins act to keep the process from going awry, despite randomevents that can cause interruption and failures. Discovering the exact repairmechanisms that help keep DNA intact during replication may help us understandthe mechanisms of tumor growth, as well as develop strategies to detect ortreat cancer.

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