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DNA replication and combination

DNA replication and combination


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"Each gamete is genetically unique because the DNA of the parent cell is shuffled before the cell divides. This helps ensure that the new organisms formed as a result of sexual reproduction are also unique."

Then why do we say that the DNA of the parent influences the characteristics of the child while the DNA of the child is formed as a combination of shuffled up nitrogenous and phosphate bases?


To explain it briefly:

Lets take a human as example, you are diploid and you have a pair of 23 chromosomes (= total 46) and the sex chromosomes which I will exclude for this explanation.

Your gamete is haploid and has therefore only one of the two paired chromosomes. So for every chromosome pair there are 2 possible chromosomes. So in total you have 223 possibilites to arrange chromosomes. The gamete from the sexual mating partner also has 223 possibilites to arrange chromosomes. So in total a new diploid organism has (223)x(223) possibilites. So unique.

However, it is always the chromosomes of the parents. So yes, parents influence the children because the genetic information of the children can be found in one of the two parents.

Furthermore you have recombination, but you already asked another question about this. So I will not expand this answer.

There are relevant articles in Wikipedia on genetic recombination and meiosis that I would recommend.


Parents influens children's characters because the shuffeling (crossing over) changes only few genes present on the parent chromosome and also these changed gene peices are also parental so actually we recieve a non parental chromosome (generated by shuffling)but the genes present on it are still parental… Hope this helps


MCAT Biology : DNA Replication and Repair

A culture of human tissue is being grown in a lab to study mitosis. A solution containing radioactively labelled cytosines was added to the culture in the middle of prophase, and then growth was halted at the end of telophase. Where would the scientists see radioactively labelled DNA?

In the mother cells only—not in the cells produced at the end of telophase

In the nuclei of every cell

Only in the nuclei of half of the cells

In the cells produced at the end of telophase—only the daughter cells

DNA is replicated in S phase. Prophase is a part of mitosis, or M phase. Since all of the DNA that would be present at the end of telophase had already been synthesized in S phase, none of the radioactively labelled cytosines would be incorporated into the DNA of any cells in the culture.

Example Question #2 : Dna Replication And Repair

Which answer choice correctly matches the molecule with its function in DNA replication?

Topoisomerase—prevents reannealing of DNA during replication

Single-stranded binding proteins—untangles supercoils

Single-stranded binding proteins—prevents reannealing of DNA during replication

Polymerase—adds nucleotides to new strands

DNase—adds nucleotides to new strands

Single-stranded binding proteins—untangles supercoils

Polymerase—adds RNA primers prior to replication

Primase—adds nucleotides to new strands

Single-stranded binding proteins—prevents reannealing of DNA during replication

Topoisomerase functions to untangle the supercoiling of DNA, which is when DNA overwinds into itself. This mechanism facilitates the unwinding action of helicase during replication. Single-stranded binding proteins bind to the two unzipped DNA strands to prevent them from prematurely coming back together into a whole molecule otherwise replication would be interrupted.

The other proteins discussed serve the following functions.

Polymerase—adds nucleotides to new strands

Primase—adds RNA primers prior to replication

DNase—cleaves and degrades DNA molecules

Example Question #1 : Dna Replication And Repair

Which of the following is the first to act during DNA replication?

Helicase is the first component of the DNA replication machinery to act during replication. It works by "unzipping" the double-stranded DNA so that replication can subsequently occur. Following the work of helicase, primase creates a primer to which the DNA polymerase will subsequently add deoxynucleotides and elongate the strand. DNA ligase acts at the end of replication by joining together the Okazaki fragments of the lagging strand.

Example Question #4 : Dna Replication And Repair

The Meselson-Stahl experiment provided the necessary evidence to discover the mechanism by which DNA replicates. They accomplished this discovery by first culturing DNA with the heavy 15 N nitrogen isotope. They then allowed the "heavy" DNA to replicate with DNA grown in normal 14 N nitrogen. The density of each generation of replicated DNA was recorded by marking its position in a test tube after centrifugation. The position of each generation was then compared to the positions of pure 15 N DNA and pure 14 N DNA.

Suppose that the first generation after replication revealed two bands after being centrifuged: one at the pure 14 N mark, and one at the pure 15 N mark. Which method of replication would this observation support?

Another generation would be needed in order to find a viable mechanism

Conservative replication proposes that both strands of DNA act as the template, but do not separate during replication. If the heavy strands were to stay together, we would expect to see a "heavy" set of DNA at the 15 N mark as well as a "normal" set of DNA at the 14 N mark.

Both semiconservative and dispersive replication would predict a singular band of DNA in between the two marks.

Example Question #5 : Dna Replication And Repair

Compared to RNA polymerase, DNA polymerase has a much lower error rate for nucleotide incorporation. What structural difference between the two polymerases accounts for this?

RNA is much less stable that DNA, and this instability makes it much harder for RNA polymerase to proofread as it incorporates bases into the sequence.

RNA molecules are proofread after they are synthesized, whereas DNA molecules are not.

DNA polymerase contains a proof-reading domain that allows it to recognize incorrect base-pair insertion before moving on RNA polymerase does not.

RNA polymerase incorporates the nucleic acids into sequences in such a way that they are more tightly bound to their partner nucleic acid, thus making it much more difficult to replace incorrect insertions.

DNA polymerase contains a proof-reading domain that allows it to recognize incorrect base-pair insertion before moving on RNA polymerase does not.

RNA polymerase does not contain a proof reading domain, making it much more error prone than DNA polymerase. This domain in DNA polymerase prevents incorrect nucleotide insertion, reducing the errors made in DNA replication.

Example Question #1 : Dna Replication And Repair

Several enzymes are required for DNA replication. What is the class of enzymes that is required for unwinding the DNA at the replication fork?

DNA helicases use ATP to break the hydrogen bonds that separate complementary strands of DNA. During DNA replication, DNA helicases move along the DNA backbone with the replication fork and are responsble for unwinding the DNA at the fork.

Example Question #1 : Dna Replication And Repair

Prions are the suspected cause of a wide variety of neurodegenerative diseases in mammals. According to prevailing theory, prions are infectious particles made only of protein and found in high concentrations in the brains of infected animals. All mammals produce normal prion protein, PrP C , a transmembrane protein whose function remains unclear.

Infectious prions, PrP Res , induce conformational changes in the existing PrP C proteins according to the following reaction:

PrP C + PrP Res → PrP Res + PrP Res

The PrP Res is then suspected to accumulate in the nervous tissue of infected patients and cause disease. This model of transmission generates replicated proteins, but does so bypassing the standard model of the central dogma of molecular biology. Transcription and translation apparently do not play a role in this replication process.

This theory is a major departure from previously established biological dogma. A scientist decides to test the protein-only theory of prion propagation. He establishes his experiment as follows:

Homogenized brain matter of infected rabbits is injected into the brains of healthy rabbits, as per the following table:

Rabbit 1 and 2: injected with normal saline on days 1 and 2

The above trials serve as controls.

Rabbit 3 and 4: injected with homogenized brain matter on days 1 and 2

The above trials use unmodified brain matter.

Rabbit 5 and 6: injected with irradiated homogenized brain matter on days 1 and 2

The above trials use brain matter that has been irradiated to destroy nucleic acids in the homogenate.

Rabbit 7 and 8: injected with protein-free centrifuged homogenized brain matter on days 1 and 2

The above trials use brain matter that has been centrifuged to generate a protein-free homogenate and a protein-rich homogenate based on molecular weight.

Rabbit 9 and 10: injected with boiled homogenized brain matter on days 1 and 2

The above trials use brain matter that have been boiled to destroy any bacterial contaminants in the homogenate.

In the material used with Rabbits 5 and 6, irradiation was used to destroy DNA. In functioning, normal cells, what types of genes typically code for DNA repair proteins?

Tumor suppresor genes, like p53 and Rb, usually code for DNA repair enzymes. Proto-oncogenes typically code for cell growth factors or receptors, and pro-apoptotic proteins would not lead to DNA repair, but would prevent tumor development via cell death pathways.

Example Question #8 : Dna Replication And Repair

DNA replication is much more accurate than RNA transcription. In replication, only one base in every ten billion, on average, is inaccurately placed.

What is the primary reason that transcription results in more errors than DNA replication?

Transcription proceeds much more quickly than replication. This results in more mistakes by RNA polymerase.

DNA polymerase synthesizes a new strand. Immediately after, a proofreading enzyme attaches and "checks" the new strand for errors.

DNA polymerase is able to repair mismatched nucleotides.

Replication is done very slowly, only a couple base pairs per second, in order to prevent mistakes by DNA polymerase.

DNA polymerase is able to repair mismatched nucleotides.

In addition to creating a new DNA strand, DNA polymerase can function as an exonuclease. DNA polymerase I has the ability to remove mismatched nucleotides from the new strand and correct them. As a result, DNA replication is very accurate, because DNA polymerase has a proofreading mechanism.

Example Question #9 : Dna Replication And Repair

Which statement best describes the function of the enzyme DNA helicase?

It proofreads the newly synthesized DNA for errors.

It begins replication of the leading strand during DNA synthesis.

It opens and unwinds the DNA double helix by disrupting the hydrogen bonds.

It prevents excess twisting of the DNA during replication.

It links nucleotide subunits together.

It opens and unwinds the DNA double helix by disrupting the hydrogen bonds.

DNA helicases are enzymes that separate the two DNA strands, and unwinds them as it progresses along the helix. It functions much like a zipper unwinding the DNA.

Example Question #10 : Dna Replication And Repair

Which base pair requires the least amount of energy to break?

The adenine and thymine base pairing forms 2 hydrogen bonds. Both cytosine and guanine form three hydrogen bonds. Thus the A-T base pair has the weakest interaction, and requires the least amount of energy to break.

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References

Watson, J. D. & Crick, F. H. C. A structure for deoxyribose nucleic acid. Nature 171, 737–738. (1953).

Crick, F. H. C. The biological replication of macromolecules. Symp. Soc. Exp. Biol. 12, 138–163 (1958).

Doty, P. Inside Nucleic Acids (Harvey Lecture, 1960) (Academic, New York, 1961).

Marmur, J. & Doty, P. Thermal renaturation of deoxyribonucleic acids. J. Mol. Biol. 3, 585–594 (1961).

Cairns, J. The bacterial chromosome and its manner of replication as seen by autoradiography. J. Mol. Biol. 6, 208–213 (1963).

Kavenoff, R., Klotz, L. C. & Zimm, B. H. On the nature of chromosome-sized DNA molecules. Cold Spring Harb. Symp. Quant. Biol. 38, 1–8 (1974).

Watson, J. D. & Crick, F. H. C. Genetical implications of the structure of deoxyribonucleic acid. Nature 171, 964–967 (1953).

Meselson, M. & Stahl, F. W. The replication of DNA in E. coli. Proc. Natl Acad. Sci. USA 44, 671–682 (1958).

Kornberg, A. Biological synthesis of DNA. Science 131, 1503–1508 (1960).

Epstein, R. H. et al. Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harb. Symp. Quant. Biol. 28, 375 (1963).

Bonhoeffer, F. & Schaller, H. A method for selective enrichment of mutants based on the high UV sensitivity of DNA containing 5-bromouracil. Biochem. Biophys. Res. Commun. 20, 93 (1965).

Kohiyama, M., Cousin, D., Ryter, A. & Jacob, F. Mutants thermosensible d'Escherichia coli K/12. I. Isolement et caracterisation rapide. Ann. Inst. Pasteur 110, 465 (1966).

Huberman, J. A., Kornberg, A. & Alberts, B. M. Stimulation of T4 bacteriophage DNA polymerase by the protein product of T4 gene 32. J. Mol. Biol. 62, 39–52 (1971).

Morris, C. F., Sinha, N. K. & Alberts, B. M. Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template. Proc. Natl Acad. Sci. USA 72, 4800–4804 (1975).

Kornberg, A. & Baker, T. A. DNA Replication 2nd edn (Freeman, New York, 1992).

Okazaki R. et al. Mechanism of DNA chain growth: possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Natl Acad. Sci. USA 59, 598–605 (1968).

Radding, C. M. Recombination activities of E. coli RecA protein. Cell 25, 3–4 (1981).

Davey, M. J. & O'Donnell, M. Mechanisms of DNA replication. Curr. Opin. Chem. Biol. 4, 581–586 (2000).

Waga, S. & Stillman, B. The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67, 721–751 (1998).

Benkovic, S. J., Valentine, A. M. & Salinas F. Replisome-mediated DNA replication. Annu. Rev. Biochem. 70, 181–208 (2001).

Alberts, B. M. The DNA enzymology of protein machines. Cold Spring Harb. Symp. Quant. Biol. 49, 1–12 (1984).

Alberts, B. The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294 (1998).

Radding, C. Colloquium introduction. Links between recombination and replication: vital roles of recombination. Proc. Natl Acad. Sci. USA 98, 8172 (2001).

Dwight, S. S. et al. Saccharomyces Genome Database (SGD) provides secondary gene annotation using the Gene Ontology (GO). Nucleic Acids Res. 30, 69–72 (2002).

Trakselis, M. A. & Benkovic, S. J. Intricacies in ATP-dependent clamp loading: variations across replication systems. Structure 9, 999–1004 (2001).

National Research Council. Bio2010: Undergraduate Education to Prepare Biomedical Research Scientists (The National Academies Press, Washington DC, 2002).

Alberts, B. et al. Molecular Biology of the Cell 4th edn (Garland, New York, 2002).


Learning Overview &mdash

Big Concepts

Faithful replication of the genetic material (DNA) is the foundation of all life on earth. The experiment by Meselson and Stahl established that DNA replicates through a semi-conservative mechanism, as predicted by Watson and Crick, in which each strand of the double helix acts as a template for a new strand with which it remains associated, until the next replication.

Bio-Dictionary Terms Used

Bacteriophage (phage) , base , base pairing , chromosome , DNA , Hershey–Chase experiment, eukaryote , mutation , nucleotides , Prokaryote (bacteria) , recombination , RNA , ultraviolet light

Terms and Concepts Explained

Equilibrium density-gradient centrifugation, DNA replication , isotope, semi-conservative DNA replication

Introduction

Matthew Meselson and Franklin Stahl (both 24 years old) met at the Marine Biological Laboratory in Woods Hole in Massachusetts and decided to test the Watson–Crick model for DNA replication, which was unproven at the time.

What Events Preceded the Experiment?

Watson and Crick proposed a "Semi-Conservative" model for DNA replication in 1953, which derived from their model of the DNA double helix. In this proposal, the strands of the duplex separate and each strand serves as a template for the synthesis of a new complementary strand. Watson's and Crick's idea for DNA replication was a model, and they did not have data to support it. Some prominent scientists had doubts.

Two other models, "Conservative" and "Dispersive", for DNA replication were proposed.

Setting Up the Experiment

A method was needed to detect a difference between the parental and daughter (newly replicated) DNA strands. Then, one could follow the parent DNA molecule in the progeny. Meselson thought to distinguish between parental and newly synthesized DNA using a density difference in the building blocks (nucleotides) used to construct the DNA. The three models for DNA replication would predict different outcomes for the density of the replicated DNA in the first- and second-generation daughter cells.

The general experimental idea was first to grow bacteria in a chemical medium to make high-density DNA and then abruptly shift the bacteria to a low-density medium so that the bacteria would now synthesize lower density DNA during upcoming rounds of replication. The old and newly synthesized DNA would be distinguished by their density.

To measure a density difference in the DNA, Meselson and Stahl invented a method called equilibrium density gradient centrifugation. In this method, the DNA is centrifuged in a tube with a solution of cesium chloride. When centrifuged, the cesium chloride, being denser than water, forms a density gradient, reaching a stable equilibrium after a few hours. The DNA migrates to a point in the gradient where its density matches the density of the CsCl solution. Heavy and light DNA would come to different resting points and thus physically separated.

Doing the Key Experiment

Meselson and Stahl first decided to study the replication of DNA from a bacteriophage, a virus that replicates inside of bacteria, and used a density difference between two forms of the nucleobase thymine (normal thymine and 5-bromouracil). These experiments did not work.

The investigators changed their plans. They studied replication of the bacterial genome and used two isotopes of nitrogen (15N (heavy) and 14N (light)) to mark the parental and newly synthesized DNA.

When the population of bacteria doubled, Meselson and Stahl noted that the DNA was of an intermediate density, half-way between the dense and light DNA in the gradient. After two doublings, half of the DNA was fully light and the other half was of intermediate density. These results were predicted by the Semi-Conservative Model and are inconsistent with the Conservative and Dispersive Models.

Meselson and Stahl did another experiment in which they used heat to separate the two strands of the daughter DNA after one round of replication. They found that one strand was all heavy DNA and the other all light. This result was consistent with the Semi-Conservative model and provided additional evidence against the Dispersive Model.

Overall, the results provided proof of Semi-Conservative replication, consistent with the model proposed by Watson and Crick.

What Happened Next?

Within a couple of weeks after their key experiment, Meselson wrote a letter to Jim Watson to share news of their result (letter included).

Max Delbruck, the Caltech physicist and biologist who had proposed the dispersive model, was elated by the results, even though Meselson and Stahl disproved his replication hypothesis, and urged the young scientists to write up their results for publication and announce the important result to the world (1958).

Scientists now know a great deal about the protein machinery responsible for DNA replication.

Closing Thoughts

The Meselson–Stahl experiment had a powerful psychological effect on the field of genetics and molecular biology. It was the first experimental test of the Watson and Crick model, and the results clearly showed that DNA was behaving in cells exactly as Watson and Crick predicted.

In addition to having a good idea, the behind-the-scenes tour of the Meselson–Stahl experiment reveals that friendship and persistence in overcoming initial failures play important roles in the scientific discovery process. Also important was an atmosphere of freedom that allowed Meselson and Stahl, then very junior, to pursue their own ideas.

Guided Paper

Meselson, M. and Stahl, F.W. (1958). The replication of DNA in Escherichia coli. Proceedings of the National Academy of Sciences U.S.A., 44: 672–682.


Discussion

Here, we assayed the function of the BLM homolog OsRecQl4 during DNA replication in rice via knockout mutants. Using the comet assay, we demonstrated induction of DSBs in aphidicolin-treated osrecql4 mutant cells. TUNEL analysis suggested that DNA damage including DSBs was induced at the RAM. The HR assay using the GUS recombination reporter showed that at least a fraction of the DSBs could be repaired by HR, explaining why HR is enhanced in osrecql4 mutants. The PI staining assay showed that un-repaired DSBs induced cell death at the root meristem. The combined results of comet, TUNEL and HR assays as well as PI staining using mutant plants under a combination of aphidicolin treatments clearly demonstrated the important role of OsRecQl4 in the process of recovery from DNA replication arrest, and also the conserved role of BLM orthologous proteins in this process. In this study, we focused on the role of OsRecQl4 in genomic maintenance during DNA replication in the RAM. However, OsRecQl4 is also expressed in the SAM (Figure 1B, C). Thus, we consider OsRecQl4 to be involved in the maintenance of genome stability during DNA replication at the SAM as well as the RAM. Therefore, increased mutations could accumulate in osrecql4 mutant plants during the mitotic cell cycle, and these mutations should be inherited by subsequent generations.

The osrecql4-1 (T-DNA line) has only a small amount of truncated protein and osrecql4-2 (Tos17 line) has a longer protein according to northern blot analysis (Figure 1B). Thus, we concluded that both the osrecql4-1 and the osrecql4-2 mutants produce an aberrant size of OsRecQl4 protein defective in the consensus domain RQC if expressed. The two mutants might have different effects. However, we revealed both mutants have hyper-sensitivity to DNA damage agents and increase cell death upon aphidicolin treatment.

In this study, we have shown that DNA replication arrest leads to a hyper-recombination phenotype in plants. Urawa et al. [39] reported that the non-transcribed spacer (NTS) between ribosomal RNA genes (rDNA), which contains a replication fork barrier of rDNA [40], enhances HR in Arabidopsis. In Escherichia coli, it has been hypothesized that a damaged DNA replication fork might be restarted by Holliday junction formation, leading to DNA cleavage by Holliday junction dissolution and finally to repair by HR. However, this mechanism runs the risk of inappropriate recombination. Thus, a stalled fork (not leading to DNA cleavage) might be processed by DNA helicases to avoid replication errors [41].

Recently, Schuermann et al. [42] reported that a defect in DNA polymerase delta 1 (POLδ1) in Arabidopsis exhibited elevated HR frequency at stalled and collapsed replication forks. This report also supported our conclusion that DNA replication stress induces HR in plants. However, the molecular mechanism connecting DNA replication stress and enhanced HR remains obscure in plants. Here, we see a relationship between accumulated DSBs, HR and cell death accompanying DNA replication arrest at the site of the meristem.

Rad51-dependent repair HR includes break-induced replication (BIR), double-Holliday junction (dHJ), and SDSA. Rad51-independent SSA also repairs DSB [43]. It was been reported recently that, with a direct repeat GU-US recombination reporter, a functional GUS gene can be generated mainly by the SSA pathway, with SDSA playing only a minor role following DSB [44]. However, during replication, induced DSBs do not induce two free ends, but rather a one-ended DSB, i.e., a DNA double-strand end (DSE). The DSE invades its sister chromatid to be repaired, with DNA synthesis by BIR [45, 46]. Thus, although increased HR efficiency was demonstrated here using the direct repeat GU-US recombination reporter to associate OsRecQl4 and HR, the HR detected should be Rad51-dependent HR repair. In future, this could be confirmed using a double mutant with Rad51. Further points in support of the hypothesis of alternative repair of the GU-US reporter are the differences in tissues (plant vs. calli) and induction (I-SceI vs. aphidicolin), which might lead to different prevalence of cell-cycle states in which the repair takes place.

We analyzed the sensitivity of osrecql4 mutants to aphidicolin and bleomycin. Since defects in OsRecQl4 enhance the sensitivity of rice to both these latter compounds, OsRecQl4 might be involved in recovery from DNA replication arrest and DSB repair. On the other hand, osrecql4 mutants showed normal growth and were fertile, although osrecql4 mutants showed aberrantly sized OsRecQl4 protein, indicating that OsRecQl4 might not be essential. In this respect, it has been reported that members of RecQ family genes in rice—OsRecQ1, OsRecQ2, OsRecQsim and OsRecQ886—are expressed in meristems [27], suggesting that RecQ helicase family members might play overlapping roles in maintaining genome stability in proliferative cells. However, homozygous blm mice exhibit growth retardation [47]. It might be interesting to evaluate growth of the osrecql4 mutant under conditions of elevated UV, since DNA replication arrest is induced by UV photoproducts.

Although the sensitivity of Arabidopsis atrecq4A mutants to bleomycin treatment was the same as that of WT plants, in our study we observed enhanced bleomycin sensitivity in rice osrecql4 mutants compared to WT plants. This might be due to differences in the system, tissue and experimental procedure. Furthermore, we observed cell death in the root meristem of osrecql4 mutant plants. These differences might be attributed to the occurrence of endoreduplication in Arabidopsis, since cells undergoing DNA damage can enter into endocycle and be separated from the mitotic cell cycle in Arabidopsis.

Our results indicate that OsRecQl4 is expressed at SAM and RAM, i.e., sites of cell division. Furthermore, expression of OsRecQl4 was induced by aphidicolin treatment but not by bleomycin (Additional file 1: Figure S5). These results suggest that, according to their level of transcription, OsRecQl4 plays a more important role in repair during DNA replication than in DSBs repair. BLM assembles at stalled replication forks [4, 6], which supports the notion of DNA replication arrest inducible expression of OsRecQl4 (Additional file 1: Figure S5). These results suggest that OsRecQl4 is required for recovery from DNA replication arrest. The osrecql4-2 mutant failed to recover from DNA replication arrest this resulted in an increased number of DSBs requiring repair, possibly by HR.

Interestingly, we found that, without aphidicolin treatment, both WT and osrecql4-2 mutants produced a very low level of DSBs (Figure 4) judging from the comet assay. Similarly, DNA damage in osrecql4-2 at the cell division root zone analyzed by TUNEL was almost comparable to that of WT plants. However, the osrecql4-2 mutant showed a greater frequency of HR than did WT plants under normal growth conditions without aphidicolin treatment. This suggested that DSBs induced in the osrecql4-2 mutant could be repaired by DNA repair systems, including HR, under normal growth conditions, since HR can proceed slowly without RecQ helicase [48]. However, excess DSBs induced by aphidicolin treatment could not be repaired by intrinsic DNA repair systems, and un-repaired DSBs were thus detected by comet and TUNEL assay. Furthermore, un-repaired DSBs might induce cell death in osrecql4 mutants upon aphidicolin treatment. Thus, OsRecQl4 might be required to rescue plants from genotoxic stress.

Broadly speaking, RecQ helicases maintain DNA stability. The RecQ helicase BLM promotes not only recovery from DNA replication arrest, but also exonuclease 1-mediated DNA resection during the initial step of DSB repair [49]. OsRecQl4 also plays a role in promoting processing of HR-mediated DSB repair in rice [26]. OsRecQl4 was expressed exclusively in meristems also, other rice RecQ-like genes were expressed in meristematic tissues [27]. Thus, we anticipate overlapping and distinct roles for the seven RecQ helicase genes in rice.


PriA: at the crossroads of DNA replication and recombination

PriA is a single-stranded DNA-dependent ATPase, DNA translocase, and DNA helicase that was discovered originally because of its requirement in vitro for the conversion of bacteriophage phi X174 viral DNA to the duplex replicative form. Studies demonstrated that PriA catalyzes the assembly of a primosome, a multiprotein complex that primes DNA synthesis, on phi X174 DNA. The primosome was shown to be capable of providing both the DNA unwinding function and the Okazaki fragment priming function required for replication fork progression. However, whereas seven proteins, PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG, were required for primosome assembly on phi X174 DNA, only DnaB, DnaC, and DnaG were required for replication from oriC, suggesting that the other proteins were not involved in chromosomal replication. Strains carrying priA null mutations, however, were constitutively induced for the SOS response, and were defective in homologous recombination, repair of UV-damaged DNA, and double-strand breaks, and both induced and constitutive stable DNA replication. The basis for this phenotype can now be explained by the ability of PriA to load replication forks at a D loop, an intermediate that forms during homologous recombination, double-strand break-repair, and stable DNA replication. Thus, a long-theorized connection between recombination and replication is demonstrated.


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7.00x Introduction to Biology and 7.05x Biochemistry or similar (biochemistry, molecular biology, and genetics).

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About this course

You’re acquainted with your DNA, but did you know that your cells synthesize enough DNA during your lifetime to stretch a lightyear in length? How does the cellular machinery accomplish such a feat without making more mistakes than you can survive? Why isn’t the incidence of cancer even higher than it is? And, if the DNA in each and every cell is two meters long, how is this genetic material compacted to fit inside the cell nucleus without becoming a tangled mess?

Are you ready to go beyond the “what" of scientific information presented in textbooks and explore how scientists deduce the details of these molecular models?

Take a behind-the-scenes look at modern molecular genetics, from the classic experimental events that identified the proteins involved in DNA replication and repair to cutting-edge assays that apply the power of genome sequencing. Do you feel confident in your ability to design molecular biology experiments and interpret data from them? We've designed the problems in this course to build your experimental design and data analysis skills.

Let’s explore the limits of our current knowledge about the replication machinery and pathways that protect the fidelity of DNA synthesis. If you are up for the challenge, join us in 7.28x Part 1: DNA Replication and Repair.

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Steps in DNA Replication

The process of DNA replication is a complex one, and involves a set of proteins and enzymes that collectively assemble nucleotides in the predetermined sequence. In response to the molecular cues received during cell division, these molecules initiate DNA replication, and synthesize two new strands using the existing strands as templates. Each of the two resultant, identical DNA molecules is composed of one old and one new strand of DNA. Hence the process of DNA replication is said to be a semi-conservative one.

The series of events that occur during prokaryotic DNA replication have been explained below.

Initiation

DNA replication begins at specific site termed as origin of replication, which has a specific sequence that can be recognized by initiator proteins called DnaA. They bind to the DNA molecule at the origin sites, thus flagging it for the docking of other proteins and enzymes essential for DNA replication. An enzyme called helicase is recruited to the site for unwinding the helices into single strands.

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Helicases break the hydrogen bonds between base pairs, in an energy-dependent manner. This point or region of DNA is now known as the replication fork. Once the helices are unwound, proteins called single-strand binding proteins (SSB) bind to the unwound regions, and prevent them for annealing. The replication process thus initiates, and the replication forks proceed in two opposite directions along the DNA molecule.

Primer Synthesis

The synthesis of a new, complementary strand of DNA using the existing strand as a template is brought about by enzymes known as DNA polymerases. In addition to replication they also play an important role in DNA repair and recombination.

However, DNA polymerases cannot start DNA synthesis independently, and require and 3′ hydroxyl group to start the addition of complementary nucleotides. This is provided by an enzyme called DNA primase which is a type of DNA-dependent RNA polymerase. It synthesizes a short stretch of RNA onto the existing DNA strands. This short segment is called a primer, and comprises 9-12 nucleotides. This gives DNA polymerase the required platform to begin copying a DNA strand. Once the primers are formed on both the strands, DNA polymerases can extend these primers into new DNA strands.

The unwinding of DNA may cause supercoiling in the regions following the fork. These DNA supercoils are relaxed by specialized enzyme called topoisomerase which binds to the DNA stretch ahead of the replication fork. It creates a nick in the DNA strand in order to relieve the supercoil.

Leading Strand Synthesis

DNA polymerases can add new nucleotides only to the 3′ end of an existing strand, and hence can synthesize DNA in 5′ → 3′ direction only. But the DNA strands run in opposite directions, and hence the synthesis of DNA on one strand can occur continuously. This is known as the leading strand.

Here, DNA polymerase III (DNA pol III) recognizes the 3′ OH end of the RNA primer, and adds new complementary nucleotides. As the replication fork progresses, new nucleotides are added in a continuous manner, thus generating the new strand.

Lagging Strand Synthesis

On the opposite strand, DNA is synthesized in a discontinuous manner by generating a series small fragments of new DNA in the 5′ → 3′ direction. These fragments are called Okazaki fragments, which are later joined to form a continuous chain of nucleotides. This strand is known as the lagging strand since the process of DNA synthesis on this strand proceeds at a lower rate.

Here, the primase adds primers at several places along the unwound strand. DNA pol III extends the primer by adding new nucleotides, and falls off when it encounters the previously formed fragment. Thus, it needs to release the DNA strand, and slide further up-stream to start the extension of another RNA primer. A sliding clamp holds the DNA in its place as it moves through the replication process.

Primer Removal

Although new DNA strands have been synthesized the RNA primers present on the newly formed strands need to be replaced by DNA. This activity is performed by the enzyme DNA polymerase I (DNA pol I). It specifically removes the RNA primers via its 5′ → 3′ exonuclease activity, and replaces them with new deoxyribonucleotides by the 5′ → 3′ DNA polymerase activity.

Ligation

After primer removal is completed the lagging strand still contains gaps or nicks between the adjacent Okazaki fragments. The enzyme ligase identifies and seals these nicks by creating a phosphodiester bond between the 5′ phosphate and 3′ hydroxyl groups of adjacent fragments.

Termination

This replication machinery halts at specific termination sites which comprise a unique nucleotide sequence. This sequence is identified by specialized proteins called tus which bind onto these sites, thus physically blocking the path of helicase. When helicase encounters the tus protein it falls off along with the nearby single-strand binding proteins.

Fact File

The DNA replication process is almost error free with the help of 3′ → 5′ exonuclease activity of the DNA polymerases. DNA pol III proofreads the nucleotides being newly added to the strand. If a nucleotide has been incorrectly added, DNA pol III recognizes the error immediately, removes the incorrect base, adds the correct nucleotide, and then continues ahead.

Difference Between Prokaryotic and Eukaryotic DNA Replication

Although the basic mechanism remains the same, eukaryotic DNA replication is much more complex, and involves a higher number of proteins and enzymes. The regulatory mechanisms for DNA replication are also more evolved and intricate.

  • In prokaryotes, DNA replication is the first step of cell division. On the other hand, eukaryotic DNA replication is intricately controlled by the cell cycle regulators, and the process takes place during the ‘S’ or synthesis phase of the cell cycle.
  • Unlike prokaryotic DNA, the eukaryotic DNA is always present in combination with histone proteins that are involved in regulation of gene expression. During replication, these proteins need to be removed just before the unwinding of DNA.
  • Owing to higher genomic size and complexity of eukaryotes, several origin and termination sites for replication are present along the DNA. The region between one set of origin and termination sites is called a replication unit or replicon, within which one event of replication takes place. This enables faster and more accurate DNA replication as compared to the prokaryotic system of having a single replicon.
  • The Okazaki fragments formed in prokaryotes are longer as compared to those in eukaryotes. In Escherichia coli (E. coli) they are about 1000 to 2000 nucleotides long whereas in eukaryotes their length ranges between 100 and 200 nucleotides.
  • Another interesting difference in prokaryotic and eukaryotic DNA replication is in the termination step of replication. In prokaryotes, the two replication forks, moving in opposite directions along the circular DNA molecule, meet at the termination site, and replication halts. However, eukaryotic DNA being a linear molecule, the lagging strand is shorter than the template strand. To avoid the loss of genetic information through such shortening, chromosomal ends have a set of repetitive sequences called telomeres that comprise noncoding DNA.

Fact File

The human DNA is copied at about 50 base pairs per second. Due to initiation of replication at multiple locations, the process is completed within one hour. If this were not the case, it would take about a month to finish replicating a single chromosome!

The genes of an organism contain all the necessary information to synthesize the right molecule, in the right amounts, and at the right time. Replication is the way to ensure that this coded information is passed down to every cell of the body, and also to the successive generations. After all, as rightly pointed by Richard Dawkins:

“They are the replicators and we are their survival machines. When we have served our purpose we are cast aside. But genes are denizens of geological time: genes are forever.”

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