These two are very similar in structure, with just one difference: the second carbon of ribose bears a hydroxyl group, while the equivalent carbon of deoxyribose has a hydrogen instead. In a cell, a nucleotide about to be added to the end of a polynucleotide chain will bear a series of three phosphate groups.

When the nucleotide joins the growing DNA or RNA chain, it loses two phosphate groups. So, in a chain of DNA or RNA, each nucleotide has just one phosphate group. Polynucleotide chains. A consequence of the structure of nucleotides is that a polynucleotide chain has directionality — that is, it has two ends that are different from each other.

DNA sequences are usually written in the 5' to 3' direction, meaning that the nucleotide at the 5' end comes first and the nucleotide at the 3' end comes last. This makes a chain with each sugar joined to its neighbors by a set of bonds called a phosphodiester linkage.

Properties of DNA. Deoxyribonucleic acid, or DNA, chains are typically found in a double helix , a structure in which two matching complementary chains are stuck together, as shown in the diagram at left. The sugars and phosphates lie on the outside of the helix, forming the backbone of the DNA; this portion of the molecule is sometimes called the sugar-phosphate backbone.

The nitrogenous bases extend into the interior, like the steps of a staircase, in pairs; the bases of a pair are bound to each other by hydrogen bonds. Structural model of a DNA double helix. This is referred to as antiparallel orientation and is important for the copying of DNA.

So, can any two bases decide to get together and form a pair in the double helix? The answer is a definite no. Because of the sizes and functional groups of the bases, base pairing is highly specific: A can only pair with T, and G can only pair with C, as shown below.

This means that the two strands of a DNA double helix have a very predictable relationship to each other. This allows each base to match up with its partner:. These two strands are complementary, with each base in one sticking to its partner on the other.

The A-T pairs are connected by two hydrogen bonds, while the G-C pairs are connected by three hydrogen bonds. When two DNA sequences match in this way, such that they can stick to each other in an antiparallel fashion and form a helix, they are said to be complementary.

Hydrogen bonding between complementary bases holds DNA strands together in a double helix of antiparallel strands. Thymine forms two hydrogen bonds with adenine, and guanine forms three hydrogen bonds with cytosine. Image modified from OpenStax Biology. Properties of RNA. Ribonucleic acid RNA , unlike DNA, is usually single-stranded.

A nucleotide in an RNA chain will contain ribose the five-carbon sugar , one of the four nitrogenous bases A, U, G, or C , and a phosphate group. Here, we'll take a look at four major types of RNA: messenger RNA mRNA , ribosomal RNA rRNA , transfer RNA tRNA , and regulatory RNAs.

Messenger RNA mRNA is an intermediate between a protein-coding gene and its protein product. The transcript carries the same information as the DNA sequence of its gene. However, in the RNA molecule, the base T is replaced with U. Once an mRNA has been produced, it will associate with a ribosome, a molecular machine that specializes in assembling proteins out of amino acids.

Image of a ribosome made of proteins and rRNA bound to an mRNA, with tRNAs bringing amino acids to be added to the growing chain. The tRNA that binds, and thus the amino acid that's added, at a given moment is determined by the sequence of the mRNA that is being "read" at that time.

Image credit: OpenStax Biology. Ribosomal RNA rRNA and transfer RNA tRNA. Ribosomal RNA rRNA is a major component of ribosomes, where it helps mRNA bind in the right spot so its sequence information can be read out.

Some rRNAs also act as enzymes, meaning that they help accelerate catalyze chemical reactions — in this case, the formation of bonds that link amino acids to form a protein. RNAs that act as enzymes are known as ribozymes.

Transfer RNAs tRNAs are also involved in protein synthesis, but their job is to act as carriers — to bring amino acids to the ribosome, ensuring that the amino acid added to the chain is the one specified by the mRNA.

Transfer RNAs consist of a single strand of RNA, but this strand has complementary segments that stick together to make double-stranded regions. This base-pairing creates a complex 3D structure important to the function of the molecule.

Structure of a tRNA. The overall molecule has a shape somewhat like an L. Image modified from Protein Data Bank work of the U. Regulatory RNA miRNAs and siRNAs. Some types of non-coding RNAs RNAs that do not encode proteins help regulate the expression of other genes.

Such RNAs may be called regulatory RNAs. For example, microRNAs miRNAs and small interfering RNAs siRNAs are small regulatory RNA molecules about 22 nucleotides long. They bind to specific mRNA molecules with partly or fully complementary sequences and reduce their stability or interfere with their translation, providing a way for the cell to decrease or fine-tune levels of these mRNAs.

These are just some examples out of many types of noncoding and regulatory RNAs. Scientists are still discovering new varieties of noncoding RNA. Summary: Features of DNA and RNA. DNA RNA Function Repository of genetic information Involved in protein synthesis and gene regulation; carrier of genetic information in some viruses Sugar Deoxyribose Ribose Structure Double helix Usually single-stranded Bases C, T, A, G C, U, A, G.

Table modified from OpenStax Biology. Explore outside of Khan Academy. Do you want to learn more about nucleotide base-pairing?

Check out this scrollable interactive from LabXchange. Want to join the conversation? Log in. Sort by: Top Voted. kind of blue. Posted 8 years ago. How do mRNA and tRNA communicate with eachother during the formation of the proteins? Downvote Button navigates to signup page. Flag Button navigates to signup page.

Show preview Show formatting options Post answer. Evan Patev. mRNA is like a recipe from a cookbook; a list of ingredients to make a protein.

mRNA is a chain of nucleotides A, U, C, and G, not T since this is RNA. A group of three nucleotides is called a codon. A codon matches with three nucleotides, called an anticodon, on a single tRNA molecule while in a ribosome.

The tRNA carries an amino acid, our ingredient to make the protein. So mRNA is the recipe, tRNA matches to the recipe bringing an ingredient, and the line of ingredients become a protein. Posted 7 years ago. If A-T bonds have 2 hydrogen bonds and G-C bonds have And if this is true, are these parts AT only parts more prone to mutations?

The first part is true, T-A bonds are less stable and more likely to come apart. The A-T bond strands also signal where DNA needs to separate for commonly transcribed genes, such as the TATA Box commonly found just before the beginning of gene sequences.

I'm not sure if they are more prone to mutations though. DNA is common to all organisms, all organisms use the same 4 nitrogenous bases, A T, C G is that right? Matt B. Entirely true. Just keep in mind that, even though all life forms have DNA, not everything that has DNA is alive: viruses can have DNA but are not living.

Are all the 46 chromosomes present in a single cell? shreya punniamoorthy. In this study the interaction between PARP and the DNA polymerase α-primase tetramer has been examined.

Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template. To maintain DNA integrity in dividing cells specific biochemical pathways have evolved to accurately coordinate the cell cycle transitions; these checkpoints link completion of one phase to onset of the following phase 1 , 3.

Moreover, DNA damage arrests the cell cycle and induces a cellular response allowing DNA repair, ensuring high fidelity of genetic information transmission 2. In eukaryotes the highly conserved DNA polymerase α-primase complex is responsible for synthesis of short RNA-DNA primers essential for the initiation step of DNA replication.

It consists of four distinct subunits: p kDa is the catalytic subunit. The primase is a heterodimer of 48 kDa endowed with catalytic activity; p58 58 kDa bears a stimulatory function, p68 68 kDa has a tethering function between p and the primase 4 , 5.

Components of the replication apparatus may act as sensors of DNA damage to stall replication forks, inducing transcription of DNA damage-inducible genes 6. A defect in the mammalian tumour suppressor gene p53 abrogates G1 arrest in response to ionizing radiation 7 , 8 by transcriptional activation of genes like GADD45 and p21 WAF1 , a cyclin-dependent kinase inhibitor 9— Furthermore, DNA damage sensors may also transduce the stress signal.

ATM, which is mutated in patients with the heritable disorder ataxia telangectasia AT , induces signalling through multiple pathways, thereby coordinating acute phase stress responses with cell cycle checkpoint control and repair of ionizing radiation and oxidative damage 12 , Patients harbouring mutations in p53 or ATM are cancer prone, implicating checkpoint controls in the prevention of genetic instability.

In yeast the catalytic subunit of DNA primase is thought to link the DNA damage response to DNA replication, whilst mutations in the PRIl gene failed to delay bud emergence in response to UV irradiation in G1 Interestingly, adjacent to the location of checkpoint-deficient mutations DNA polymerase ε encompasses a zinc finger resembling the zinc fingers of PARP involved in binding to single-strand breaks 15 , suggesting that both proteins recognize a similar structure in DNA.

Poly ADP-ribose polymerase is a component of the immediate cellular response to genotoxic stress, playing a critical role in cell recovery from DNA damage 16 , Purified PARP was shown to suppress in vitro replication of SV40 DNA 18 and to inhibit DNA replication by human replicative DNA polymerases α, δ and ε In contrast, Simbulan et al.

demonstrated that in vitro PARP stimulated DNA polymerase α through a physical association The same authors demonstrated that the PARP-DNA polymerase α association was required in differentiation-linked DNA replication 21 , Furthermore, in mice lacking PARP proliferating cells are exquisitely sensitive to DNA damaging agents compared with wild-type cells, as measured by: i apoptotic cell death of splenocytes exposed to N -methyl- N -nitrosourea MNU ; ii necrosis of the epithelial cells of the small intestine located within the crypts, causing death of PARP-deficient mice by 3 days following 8 Gy γ-irradiation All these data strongly suggest that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

In this work we present evidence that PARP and DNA polymerase α-primase are physically associated in dividing cells, permitting coordination of the initiation of DNA replication with the resolution of replication blocks induced by DNA strand breaks.

Primary fibroblasts MEFs were isolated from HeLa cells 5 × 10 6 were washed twice with phosphate-buffered saline PBS and lysed on ice in 1 ml lysis buffer 20 mM Tris-HCl, pH 8. Solubilized cell lysates µg protein , precleared for 16 h with 15 µl protein A-Sepharose beads Pharmacia Biotech , were incubated for 2 h at 4°C with either monoclonal antibodies [anti-DNA polymerase α-primase antibodies SJK 30 , provided by M.

Smulson, Georgetown University School of Medicine, Washington, and SJK 30 , provided by J. Hurwitz, Sloan-Kettering Cancer Center, New York; anti-β-galactosidase clone GAL13, Sigma], a polyclonal anti-PARP antibody or a pre-immune serum.

Immunocomplexes were precipitated by addition of 30 µl protein A-Sepharose beads and washed five times in 20 mM Tris-HCl, pH 8. Proteins were transferred onto nitrocellulose and immunoblotted with appropriate antibodies.

Immunoblotting was performed with an enhanced chemiluminescence detection system Amersham. In in vitro experiments 1 µg purified DNA polymerase α-primase and the indicated domains of PARP 1 µg each were pre-incubated in µl lysis buffer for 1 h on ice and immunoprecipitation was performed as described above using the polyclonal anti-PARP antibody or the pre-immune serum as control.

Cells fixed on coverslips were incubated for 16 h with the first antibodies diluted in PBS, 0. Weiss, ESBS, Illkirch, France.

After three washes with PBS containing 0. DNA polymerase activity was tested in 60 µl buffer containing 10 mM Tris-HCl, pH 7.

Incorporation of radiolabelled nucleotides was determined by TCA precipitation. DNA polymerase α-primase inhibition was performed in the presence of 10 µl SJK or an antibody directed against the DNA polymerase α 68 kDa subunit.

After renaturation for 16 h at 4°C in 10 ml buffer containing 10 mM Tris-HCl, pH 7. The membrane was then washed twice in 10 mM Tris-HCl, pH 8.

Cells were released from the cell cycle block by washing three times with PBS and adding fresh complete medium. At various times after release from the aphidicolin block samples were harvested for immunoprecipitation and flow cytometry analysis using an Epics Elite Coulter.

Mouse embryonic fibroblasts were synchronized in G0 in DMEM containing 0. Cells were harvested, treated or mock-treated with µM MMS for 30 min at 37°C and released into fresh complete medium.

Twenty four hours later cells were pulse labelled with 10 µM 5-bromodeoxyuridine BrdU for 1 h and the percentage of cells in S phase was monitored as described Previous studies 20 have shown that PARP is physically associated in vitro with DNA polymerase α. To assess the existence of this association in living cells HeLa whole cell extracts were immunoprecipitated using two different monoclonal antibodies raised against the DNA polymerase α-primase kDa subunit 30 and the immune complex was subjected to SDS-PAGE.

Figure 1A shows that PARP kDa , whose activity was detected by activity blot 31 , was immunoprecipitated with a polyclonal anti-PARP antibody lane 1 and was also specifically co-immunoprecipitated with DNA polymerase α-primase using anti-DNA polymerase α antibodies lanes 2 and 3 but not with an anti-β-galactosidase antibody as a negative control lane 4.

Conversely, HeLa whole cell extracts were immunoprecipitated with a polyclonal anti-PARP antibody and the immune complex was assayed for DNA polymerase activity using DNase I-activated DNA as a substrate Fig. As expected, DNA polymerase activity was co-immunoprecipitated with PARP.

In control experiments DNA polymerase activity was not associated either with the pre-immune serum or with protein A-Sepharose beads. Furthermore, both enzymes were co-immunoprecipitated by specific antibodies to either PARP or DNA polymerase α data not shown from lysates of insect cells co-infected with recombinant baculovirus expressing both the p large subunit of DNA polymerase α 32 , 33 and PARP To exclude the possibility that this association could occur via tightly bound DNA fragments rather than by specific protein-protein interactions the immune complex was assayed for DNA polymerase activity in the absence or presence of DNase I-activated DNA as substrate.

Under the same experimental conditions PARP was found associated with DNA polymerase β only, in keeping with its potential role in base excision repair BER PARP interacts with DNA polymerase α-primase.

A HeLa cell extracts were subjected to immunoprecipitation with anti-PARP lane 1 or two different anti-DNA polymerase α-primase antibodies SJK, lane 2; SJK, lane 3.

An anti-β-galactosidase antibody was used as a negative control lane 4. Immunoprecipitates were then analysed by immunoblotting using an anti-PARP antibody top. Immunoprecipitates were also analysed by activity blot bottom.

Figure 2 shows typical PARP and DNA polymerase patterns of doubly stained nuclei observed in proliferating HeLa cells. Panels A and B show confocal images of DNA polymerase α-primase and PARP labelling respectively within the same nucleus.

Co-localization yellow is observed at the nuclear periphery and in nucleoli; both patterns overlapped within the limits of the procedure. Altogether, these results indicate that in vivo the two proteins are in close vicinity and are both preferentially present in the nuclear envelope.

Confocal analysis of PARP and DNA polymerase α double staining in HeLa cells. A Rhodamine-labelled DNA polymerase α red ; B fluorescein-labelled PARP green ; C merged image regions of overlap are in yellow. The interacting domains between PARP and DNA polymerase α-primase were mapped using two independent approaches.

First, equimolar amounts of purified homogeneous human DNA polymerase α-primase and full-length PARP or PARP functional domains 29 kDa DBD or 40 kDa catalytic domain; Fig.

Then an anti-PARP antibody or the pre-immune serum was added to the reaction mixture and the immunoprecipitates were assayed for DNA polymerase α activity. As shown in Figure 3B , DNA polymerase activity co-immunoprecipitated with full-length PARP as well as with the 29 kDa DBD, but not with the 40 kDa catalytic domain or the pre-immune serum.

To determine whether this association also exists in vivo , in a second approach HeLa H cells constitutively expressing the human PARP 29 kDa domain as well as the parental cell line HpECV were used The proteins were identified by Western blot analysis using monoclonal anti-PARP antibody C1,9 17; Fig.

Lane 4 shows the typical pattern of cell line H expressing the recombinant 29 kDa DBD and also containing full-length endogenous PARP. Both proteins were immunoprecipitated with anti-PARP antibody lane 3 and with SJK lane 2 , but not with a non-specific anti-β-galactosidase antibody lane 1.

PARP was immunoprecipitated with SJK in HeLa cells, as already reported in Figure 1A. Thus both in vitro and in vivo PARP contacts DNA polymerase α-primase through its 29 kDa DBD. In crude lysates obtained from the parental line HpECV no protein migrating at a molecular weight of 29 kDa was co-immunoprecipitated Fig.

To assess the integrity of the PARP zinc fingers in this interaction 35 S-radiolabelled PARP functional domains were synthesized in vitro and used in Far western blot analysis. Purified human DNA polymerase α-primase tetramer was separated by SDS-PAGE, transferred to nitrocellulose membrane and, after renaturation of the proteins, hybridized.

As shown in Figure 3D , the kDa catalytic subunit of DNA polymerase α-primase bound to the full-length PARP as well as the 46 kDa DBD, whereas the 40 kDa C-terminal catalytic domain did not interact under these conditions.

Interestingly, the two mutated forms of the 46 kDa DBD failed to interact with the kDa catalytic subunit of DNA polymerase α-primase, strongly suggesting that integrity of the second zinc finger, at least, is required.

From this Far western blot experiment we also concluded that the interaction is not mediated by DNA. p and PARP were present throughout the cell cycle Fig. Cell lysates from different stages of the cell cycle were immunoprecipitated with the anti-DNA polymerase α-primase antibody SJK The immune complex was immunoblotted with both monoclonal antibody directed against the kDa subunit of DNA polymerase α-primase and anti-PARP antibody Fig.

Although DNA polymerase α-primase was efficiently immunoprecipitated throughout the cell cycle, PARP was found associated with DNA polymerase α-primase only during the S and G2 phases of the cell cycle, and not during G1 phase. The same amount of the large subunit p of DNA polymerase α-primase was present in both extracts.

The DNA binding domain of PARP interacts with the catalytic subunit of DNA polymerase α-primase. A Modular organization of the human PARP molecule. B Purified DNA polymerase α-primase was incubated with PARP kDa or the purified domains of PARP 29 and 40 kDa.

Immunoprecipitation was then performed with anti-PARP and the precipitates were assayed for DNA polymerase α activity at 0 and 30 min. Immunoprecipitation with the pre-immune serum was performed as a negative control.

C H cell lysates were immunoprecipitated with an anti-β-galactosidase antibody as a negative control lane 1 , anti-DNA polymerase α-primase antibody SJK lane 2 or anti-PARP antibody as positive control lane 3.

Aliquots of 10 µg H crude lysate were loaded as controls lane 4. HpECV cell lysates were immunoprecipitated with anti-DNA polymerase α-primase SJK lane 5. Proteins were then analysed by immunoblotting with anti-PARP. IgG H , immunoglobulin G heavy chain; IgG L , immunoglobulin G light chain.

D Far western blotting analysis. Purified DNA polymerase α-primase was separated by SDS-PAGE, transferred to nitrocellulose membrane and hybridized with 35 S-radiolabelled full-length PARP lane 1 , wild-type 46 kDa domain lane 2 , the double point mutant C21G CG lane 3 , the single point mutant RI 19 lane 4 or the 40 kDa catalytic domain lane 5.

The involvement of PARP in progression of the replication fork following DNA damage was monitored by the ability of cells to progress into S phase after exposure to sublethal doses of MMS. Immediately following release into fresh medium some cells were exposed to MMS, whereas other cells were left untreated; 24 h later they were all pulse labelled with BrdU Fig.

Cell cycle-dependent interaction of PARP with DNA polymerase α-primase. Aphidicolin-arrested HeLa cells were released into fresh medium.

Timed samples were monitored for progression through the cell cycle by flow cytometric analysis A.

Figure 1: A comparison of the helix and base structure of RNA and DNA. Credit: Technology Networks. Z-DNA is thought to play a role in regulating gene expression and may be produced in the wake of DNA processing enzymes, like DNA polymerase. Triplex-forming oligonucleotides TFOs can bind conventional two-stranded DNA, which can help guide agents that are used to modify DNA to specific genomic locations.

H-DNA is an endogenous, triple-stranded DNA molecule that encourages mutation of the genome. What are the key differences between DNA and RNA? DNA encodes all genetic information, and is the blueprint from which all biological life is created. In the long-term, DNA is a storage device, a biological flash drive that allows the blueprint of life to be passed between generations2.

RNA functions as the reader that decodes this flash drive. This reading process is multi-step and there are specialized RNAs for each of these steps. What are the three types of RNA? Messenger RNA mRNA copies portions of genetic code, a process called transcription, and transports these copies to ribosomes, which are the cellular factories that facilitate the production of proteins from this code.

Transfer RNA tRNA is responsible for bringing amino acids, basic protein building blocks, to these protein factories, in response to the coded instructions introduced by the mRNA. This protein-building process is called translation.

Finally, Ribosomal RNA rRNA is a component of the ribosome factory itself without which protein production would not occur3. How many strands does RNA have?

Except for some viruses, RNA typically has one strand. RNA is a polymer consisting of chains of nucleotides. These are nitrogenous bases attached to phosphate groups and ribose sugars.

The four bases in RNA are adenine, uracil, cytosine and guanine. How does DNA differ from RNA? There are several differences that separate DNA from RNA.

What are the main structural differences between DNA and RNA molecules? DNA and RNA have significant structural differences. DNA is double-stranded, forming a double helix, while RNA is usually single-stranded.

The sugar in DNA is deoxyribose, whereas RNA contains ribose. Furthermore, DNA uses the bases adenine, thymine, cytosine, and guanine, while RNA uses adenine, uracil, cytosine, and guanine. How do the roles of DNA and RNA differ in protein synthesis? DNA and RNA have distinct roles in protein synthesis.

DNA holds the genetic information or "blueprint" for the protein. RNA, specifically messenger RNA mRNA , carries this information from DNA to the ribosomes, where translation into a protein sequence occurs.

Transfer RNA tRNA and ribosomal RNA rRNA also play key roles in this process. What are the stability differences between DNA and RNA and how do they affect their functions?

DNA is more stable due to its double-stranded structure and the presence of deoxyribose sugar, making it suited for long-term genetic storage. RNA, being less stable, is suitable for short-term tasks like transferring genetic information from DNA during protein synthesis.

How do DNA and RNA interact in the process of genetic information transfer? During genetic information transfer, DNA is transcribed into RNA in a process called transcription.

RNA, specifically mRNA, then carries this genetic information to the ribosomes for translation into proteins. What are some real-world applications that hinge on the differences between DNA and RNA?

Understanding the differences between DNA and RNA is crucial in various fields. For example, in biotechnology, DNA is manipulated for genetic engineering, while RNA interference is used to control gene expression. In medicine, DNA sequencing helps in diagnosing genetic disorders, and RNA vaccines like COVID mRNA vaccines have become crucial in disease prevention.

I Understand. RNA — 5 Key Differences and Comparison DNA and RNA are the two most important molecules in cell biology, but what are the key differences between them? Article Published: December 18, Last Updated: January 22, Ruairi J Mackenzie.

As senior science writer, Ruairi pens and edits scientific news, articles and features, with a focus on the complexities and curiosities of the brain and emerging informatics technologies. Learn about our editorial policies. Patients harbouring mutations in p53 or ATM are cancer prone, implicating checkpoint controls in the prevention of genetic instability.

In yeast the catalytic subunit of DNA primase is thought to link the DNA damage response to DNA replication, whilst mutations in the PRIl gene failed to delay bud emergence in response to UV irradiation in G1 Interestingly, adjacent to the location of checkpoint-deficient mutations DNA polymerase ε encompasses a zinc finger resembling the zinc fingers of PARP involved in binding to single-strand breaks 15 , suggesting that both proteins recognize a similar structure in DNA.

Poly ADP-ribose polymerase is a component of the immediate cellular response to genotoxic stress, playing a critical role in cell recovery from DNA damage 16 , Purified PARP was shown to suppress in vitro replication of SV40 DNA 18 and to inhibit DNA replication by human replicative DNA polymerases α, δ and ε In contrast, Simbulan et al.

demonstrated that in vitro PARP stimulated DNA polymerase α through a physical association The same authors demonstrated that the PARP-DNA polymerase α association was required in differentiation-linked DNA replication 21 , Furthermore, in mice lacking PARP proliferating cells are exquisitely sensitive to DNA damaging agents compared with wild-type cells, as measured by: i apoptotic cell death of splenocytes exposed to N -methyl- N -nitrosourea MNU ; ii necrosis of the epithelial cells of the small intestine located within the crypts, causing death of PARP-deficient mice by 3 days following 8 Gy γ-irradiation All these data strongly suggest that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

In this work we present evidence that PARP and DNA polymerase α-primase are physically associated in dividing cells, permitting coordination of the initiation of DNA replication with the resolution of replication blocks induced by DNA strand breaks.

Primary fibroblasts MEFs were isolated from HeLa cells 5 × 10 6 were washed twice with phosphate-buffered saline PBS and lysed on ice in 1 ml lysis buffer 20 mM Tris-HCl, pH 8.

Solubilized cell lysates µg protein , precleared for 16 h with 15 µl protein A-Sepharose beads Pharmacia Biotech , were incubated for 2 h at 4°C with either monoclonal antibodies [anti-DNA polymerase α-primase antibodies SJK 30 , provided by M.

Smulson, Georgetown University School of Medicine, Washington, and SJK 30 , provided by J. Hurwitz, Sloan-Kettering Cancer Center, New York; anti-β-galactosidase clone GAL13, Sigma], a polyclonal anti-PARP antibody or a pre-immune serum.

Immunocomplexes were precipitated by addition of 30 µl protein A-Sepharose beads and washed five times in 20 mM Tris-HCl, pH 8. Proteins were transferred onto nitrocellulose and immunoblotted with appropriate antibodies. Immunoblotting was performed with an enhanced chemiluminescence detection system Amersham.

In in vitro experiments 1 µg purified DNA polymerase α-primase and the indicated domains of PARP 1 µg each were pre-incubated in µl lysis buffer for 1 h on ice and immunoprecipitation was performed as described above using the polyclonal anti-PARP antibody or the pre-immune serum as control.

Cells fixed on coverslips were incubated for 16 h with the first antibodies diluted in PBS, 0. Weiss, ESBS, Illkirch, France. After three washes with PBS containing 0. DNA polymerase activity was tested in 60 µl buffer containing 10 mM Tris-HCl, pH 7.

Incorporation of radiolabelled nucleotides was determined by TCA precipitation. DNA polymerase α-primase inhibition was performed in the presence of 10 µl SJK or an antibody directed against the DNA polymerase α 68 kDa subunit.

After renaturation for 16 h at 4°C in 10 ml buffer containing 10 mM Tris-HCl, pH 7. The membrane was then washed twice in 10 mM Tris-HCl, pH 8. Cells were released from the cell cycle block by washing three times with PBS and adding fresh complete medium.

At various times after release from the aphidicolin block samples were harvested for immunoprecipitation and flow cytometry analysis using an Epics Elite Coulter. Mouse embryonic fibroblasts were synchronized in G0 in DMEM containing 0. Cells were harvested, treated or mock-treated with µM MMS for 30 min at 37°C and released into fresh complete medium.

Twenty four hours later cells were pulse labelled with 10 µM 5-bromodeoxyuridine BrdU for 1 h and the percentage of cells in S phase was monitored as described Previous studies 20 have shown that PARP is physically associated in vitro with DNA polymerase α.

To assess the existence of this association in living cells HeLa whole cell extracts were immunoprecipitated using two different monoclonal antibodies raised against the DNA polymerase α-primase kDa subunit 30 and the immune complex was subjected to SDS-PAGE.

Figure 1A shows that PARP kDa , whose activity was detected by activity blot 31 , was immunoprecipitated with a polyclonal anti-PARP antibody lane 1 and was also specifically co-immunoprecipitated with DNA polymerase α-primase using anti-DNA polymerase α antibodies lanes 2 and 3 but not with an anti-β-galactosidase antibody as a negative control lane 4.

Conversely, HeLa whole cell extracts were immunoprecipitated with a polyclonal anti-PARP antibody and the immune complex was assayed for DNA polymerase activity using DNase I-activated DNA as a substrate Fig.

As expected, DNA polymerase activity was co-immunoprecipitated with PARP. In control experiments DNA polymerase activity was not associated either with the pre-immune serum or with protein A-Sepharose beads.

Furthermore, both enzymes were co-immunoprecipitated by specific antibodies to either PARP or DNA polymerase α data not shown from lysates of insect cells co-infected with recombinant baculovirus expressing both the p large subunit of DNA polymerase α 32 , 33 and PARP To exclude the possibility that this association could occur via tightly bound DNA fragments rather than by specific protein-protein interactions the immune complex was assayed for DNA polymerase activity in the absence or presence of DNase I-activated DNA as substrate.

Under the same experimental conditions PARP was found associated with DNA polymerase β only, in keeping with its potential role in base excision repair BER PARP interacts with DNA polymerase α-primase.

A HeLa cell extracts were subjected to immunoprecipitation with anti-PARP lane 1 or two different anti-DNA polymerase α-primase antibodies SJK, lane 2; SJK, lane 3. An anti-β-galactosidase antibody was used as a negative control lane 4.

Immunoprecipitates were then analysed by immunoblotting using an anti-PARP antibody top. Immunoprecipitates were also analysed by activity blot bottom. Figure 2 shows typical PARP and DNA polymerase patterns of doubly stained nuclei observed in proliferating HeLa cells.

Panels A and B show confocal images of DNA polymerase α-primase and PARP labelling respectively within the same nucleus. Co-localization yellow is observed at the nuclear periphery and in nucleoli; both patterns overlapped within the limits of the procedure.

Altogether, these results indicate that in vivo the two proteins are in close vicinity and are both preferentially present in the nuclear envelope. Confocal analysis of PARP and DNA polymerase α double staining in HeLa cells. A Rhodamine-labelled DNA polymerase α red ; B fluorescein-labelled PARP green ; C merged image regions of overlap are in yellow.

The interacting domains between PARP and DNA polymerase α-primase were mapped using two independent approaches. First, equimolar amounts of purified homogeneous human DNA polymerase α-primase and full-length PARP or PARP functional domains 29 kDa DBD or 40 kDa catalytic domain; Fig.

Then an anti-PARP antibody or the pre-immune serum was added to the reaction mixture and the immunoprecipitates were assayed for DNA polymerase α activity.

As shown in Figure 3B , DNA polymerase activity co-immunoprecipitated with full-length PARP as well as with the 29 kDa DBD, but not with the 40 kDa catalytic domain or the pre-immune serum. To determine whether this association also exists in vivo , in a second approach HeLa H cells constitutively expressing the human PARP 29 kDa domain as well as the parental cell line HpECV were used The proteins were identified by Western blot analysis using monoclonal anti-PARP antibody C1,9 17; Fig.

Lane 4 shows the typical pattern of cell line H expressing the recombinant 29 kDa DBD and also containing full-length endogenous PARP.

Both proteins were immunoprecipitated with anti-PARP antibody lane 3 and with SJK lane 2 , but not with a non-specific anti-β-galactosidase antibody lane 1.

PARP was immunoprecipitated with SJK in HeLa cells, as already reported in Figure 1A. Thus both in vitro and in vivo PARP contacts DNA polymerase α-primase through its 29 kDa DBD. In crude lysates obtained from the parental line HpECV no protein migrating at a molecular weight of 29 kDa was co-immunoprecipitated Fig.

To assess the integrity of the PARP zinc fingers in this interaction 35 S-radiolabelled PARP functional domains were synthesized in vitro and used in Far western blot analysis.

Purified human DNA polymerase α-primase tetramer was separated by SDS-PAGE, transferred to nitrocellulose membrane and, after renaturation of the proteins, hybridized. As shown in Figure 3D , the kDa catalytic subunit of DNA polymerase α-primase bound to the full-length PARP as well as the 46 kDa DBD, whereas the 40 kDa C-terminal catalytic domain did not interact under these conditions.

Interestingly, the two mutated forms of the 46 kDa DBD failed to interact with the kDa catalytic subunit of DNA polymerase α-primase, strongly suggesting that integrity of the second zinc finger, at least, is required.

Ribose-seq uses the UMI in the adaptors for high-throughput sequencing which allows to perform deduplication of the sequencing reads, and thus providing for a more accurate detection of rNMP hotspots.

Ribose-seq is not specific to yeast cells; the technique could potentially be applied to any cell type from any species, making it a useful tool for elucidating the and location and pattern of rNMPs in most organisms.

Ribose-seq uses common laboratory equipment and reagents that are fairly easy to source, which makes it an accessible technique for use in many molecular biology laboratories. Because it targets single rNMPs, ribose-seq cannot capture longer ribonucleotide tracts.

Therefore, ribose-seq would not be an appropriate technique for mapping RNA primers or Okazaki fragments, which can be formed during DNA replication or breaks. Ribose-seq takes about 4 days to prepare the rNMP libraries from the genomic DNA. Ribose-Map is a bioinformatic tool for processing and analysis of large, complex rNMP raw sequencing datasets.

RESCOT is a computational method that calculates the genomic coverage of RNMP-captured regions for a given choice of restriction enzymes and then optimize the RE sets and select the RE set with highest coverage to maximize rNMP capture rate.

Contents move to sidebar hide. Article Talk. Read Edit View history. Tools Tools. What links here Related changes Upload file Special pages Permanent link Page information Cite this page Get shortened URL Download QR code Wikidata item. Download as PDF Printable version. Genetic mapping technique.

Nature Methods. doi : PMC PMID Nature Reviews Molecular Cell Biology. Molecular Cell. Bibcode : Sci Retrieved 28 Feb The findings resulted from collaboration between researchers in Storici's laboratory at the Georgia Institute of Technology -- with graduate students Kyung Duk Koh and Sathya Balachander -- and at the University of Colorado Anschutz Medical School with assistant professor Jay Hesselberth.

In García-Velasco, Juan; Seli, Emre eds. Human Reproductive Genetics. Academic Press. ISBN Proceedings of the National Academy of Sciences USA. Bibcode : PNAS EMBO Reports.

Bibcode : iSci Critical Reviews in Biochemistry and Molecular Biology. Journal of Virology. Journal of Biological Chemistry. Mol Cell. Characterization by 32P-postlabeling". Mutation Research. Bibcode : Nanos Journal of the American Chemical Society. Bibcode : PNAS.. RNA Biology.

Annual Review of Genomics and Human Genetics. Bibcode : Natur. Nature Communications. Bibcode : NatCo..

This S phase poly ADP-ribose does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly ADP-ribose more than fold, implicating unligated Okazaki fragments as the source of S phase PARP activity.

Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.

The antibody against TKT sc, dilution was purchased from Santa Cruz Biotechnology. RPIA , dilution antibody was from Abcam.

Anti-β-actin A, dilution , antibody was purchased from GeneScript. Anti-Flag M, dilution , Anti-Myc M, dilution , and anti-HA M, dilution antibodies were obtained from Abmart. DAPI D was from Sigma-Aldrich. EdU A and Azide Alexa Fluor A were purchased from Invitrogen. HEKT ATCC Number: CRL , HeLa ATCC Number: CCL-2 and MCF7 ATCC Number: HTB were purchased from Shanghai Cell Bank and tested negative for mycoplasma contamination.

HeLa cells were authenticated using Short Tandem Repeat STR analysis by Shanghai Biowing Applied Biotechnology Company.

PFKFB3 knockout HeLa cell lines are kindly provided by Dr. Ye Dan, MCB laboratory, Fudan University. The guide sequence targeting the human PFKFB3 gene is 5ʹ- AGC TGA CTC GCT ACC TCA AC-3ʹ. The TKT or TKTL1-positive stable cells were lysed on ice in 0. The precipitates were washed three times with 0.

The peptides in the supernatant were collected by centrifugation and dried in a speed vacuum Eppendorf. Samples were re-dissolved in NH 4 HCO 3 buffer containing 0.

Plasmid transfections were carried out by the Polyethylenimine PEI , Lipofectamine Invitrogen , or calcium phosphate methods. In the Lipofectamine transfection method. This is important to balance the pH for transfection efficiency. The plates were swirled and placed back into the incubator. For immunoprecipitation, cells were lysed with 0.

The binding complexes were washed with 0. For immunohistochemical staining, tissue sections were deparaffinized by xylene two times and then hydrated.

Sections were developed with the DAB kit, and the reaction was stopped with water. The H-score method, which combines the immunoreactivity intensity values and the percentage of stained tumor cells, was used to quantify the positive score of each sample.

Flag Beads Sigma was used for immunoprecipitation. Ubiquitination was analyzed by immunoblotting using anti-HA and anti-Flag antibodies. CDH1 knockdown was carried out using synthetic siRNA oligonucleotides synthesized by Genepharma.

A scrambled siRNA was used as a control. For each target gene, we employed two effective target sequences to exclude off-target effects.

Transfections were performed by using Lipofectamine Invitrogen. The knockdown efficiency was verified by q-RT-PCR or western blotting. Supplementary Table 1 lists the DNA sequences for the siRNA. To generate cells stably knocked down for TKTL1 and TKT, lentiviruses carrying pMKO empty vector, pMKO-TKTL1, or pMKO-TKT were produced in HEKT and HeLa cells, using VSVG and GAG as packaging plasmids.

Puromycin was used to select stable cells for ~5 days. The DNA sequences of the siRNAs are listed in Supplementary Table 1. For forward TKT activity measurement, we coupled measurement of enzyme activity with GAPDH enzymes.

Each experiment was repeated three times. For reverse TKT activity measurement, we measured enzyme activity through detection of either R5P for reaction 3 or E4P production for reaction 3 in reversed reactions.

Samples were added to a 1. For measuring in vivo TKT activity, cells were sonicated and centrifuged, after which the resulting supernatant was collected for analysis. The cells were next analyzed using a fluorescence-activated cell sorter FACS.

The gating strategy of flow cytometry was shown in Supplementary Fig. The wild-type TKT and TKTL1 were cloned in vector pSJ3 with 6× HIS tag at the N-terminal.

Recombinant heterodimer of TKT and TKTL1 was cloned into the pET-Duet vector. TKTL1 was fused in-frame with 6× HIS tag at the N-terminal, while TKT was inserted in the vector with a Flag tag at the C-terminal. Plasmids were transformed into E. coli BL21 DE3 pLysS strain and protein expression was induced by addition of 0.

Cells were lysed by sonication and nickel columns GE Healthcare were used to purify proteins. Heterodimer proteins were isolated by sequential affinity purification using Nickel resin followed by FLAG beads Sigma-Aldrich.

All primers for analysis were synthesized by Generay Shanghai. Primer sequences are listed in Supplementary Table 1. The analysis was performed by using an Applied Biosystems HT Sequence Detection System, with SYBR green labeling.

The mobile phase comprised eluent A 0. The elution program was as follows, 0. The flow rate of the pump was 0. Glyceraldehydep and dihydroxyacetone phosphate ions were monitored at precursor-product ; ribosep and xylulosep ions at ; sedulosephosphate ions at ; erythrosephosphate ions at ; fructosephosphate ions at Each measurement was obtained at least in triplicate.

The cells were washed twice with PBS after collection to completely remove labeled glucose that was not metabolized by the cells. M2 of R5P at , IMP at , AMP at , GMP at , and PRPP at For absolute R5P concentration measurement, 0, 5, 10, 50, , , and nmol R5P from cell lysis were used to obtain an R5P standard curve.

After R5P detection via LC-MS, the absolute concentration of R5P was calculated using the R5P standard curve. Proteins were passed over the gel filtration column.

The flow speed rate was 0. Fractions were collected every 0. Molecular mass was determined by Gel Filtration Calibration Kit HMW GE Healthcare. Cells were harvested and washed with PBS twice to remove the remaining medium. DAPI was subsequently added, for nuclear staining. Results were acquired in flow cytometer or cells were observed under a fluorescence microscope.

The binding kinetics and affinity of TKT with TKTL1 protein or small molecules were analyzed by SPR Biacore T, GE Healthcare. To determine the K d of TKT and TKTL1, TKTL1 protein was diluted to a series of concentrations starting at Statistical analysis was performed using Prism 6.

Two-tailed Student's t -test was performed for the two-group analysis. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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Smulson, Georgetown University School of Medicine, Washington, and SJK 30 , provided by J. Hurwitz, Sloan-Kettering Cancer Center, New York; anti-β-galactosidase clone GAL13, Sigma], a polyclonal anti-PARP antibody or a pre-immune serum.

Immunocomplexes were precipitated by addition of 30 µl protein A-Sepharose beads and washed five times in 20 mM Tris-HCl, pH 8.

Proteins were transferred onto nitrocellulose and immunoblotted with appropriate antibodies. Immunoblotting was performed with an enhanced chemiluminescence detection system Amersham. In in vitro experiments 1 µg purified DNA polymerase α-primase and the indicated domains of PARP 1 µg each were pre-incubated in µl lysis buffer for 1 h on ice and immunoprecipitation was performed as described above using the polyclonal anti-PARP antibody or the pre-immune serum as control.

Cells fixed on coverslips were incubated for 16 h with the first antibodies diluted in PBS, 0. Weiss, ESBS, Illkirch, France. After three washes with PBS containing 0. DNA polymerase activity was tested in 60 µl buffer containing 10 mM Tris-HCl, pH 7.

Incorporation of radiolabelled nucleotides was determined by TCA precipitation. DNA polymerase α-primase inhibition was performed in the presence of 10 µl SJK or an antibody directed against the DNA polymerase α 68 kDa subunit. After renaturation for 16 h at 4°C in 10 ml buffer containing 10 mM Tris-HCl, pH 7.

The membrane was then washed twice in 10 mM Tris-HCl, pH 8. Cells were released from the cell cycle block by washing three times with PBS and adding fresh complete medium. At various times after release from the aphidicolin block samples were harvested for immunoprecipitation and flow cytometry analysis using an Epics Elite Coulter.

Mouse embryonic fibroblasts were synchronized in G0 in DMEM containing 0. Cells were harvested, treated or mock-treated with µM MMS for 30 min at 37°C and released into fresh complete medium. Twenty four hours later cells were pulse labelled with 10 µM 5-bromodeoxyuridine BrdU for 1 h and the percentage of cells in S phase was monitored as described Previous studies 20 have shown that PARP is physically associated in vitro with DNA polymerase α.

To assess the existence of this association in living cells HeLa whole cell extracts were immunoprecipitated using two different monoclonal antibodies raised against the DNA polymerase α-primase kDa subunit 30 and the immune complex was subjected to SDS-PAGE.

Figure 1A shows that PARP kDa , whose activity was detected by activity blot 31 , was immunoprecipitated with a polyclonal anti-PARP antibody lane 1 and was also specifically co-immunoprecipitated with DNA polymerase α-primase using anti-DNA polymerase α antibodies lanes 2 and 3 but not with an anti-β-galactosidase antibody as a negative control lane 4.

Conversely, HeLa whole cell extracts were immunoprecipitated with a polyclonal anti-PARP antibody and the immune complex was assayed for DNA polymerase activity using DNase I-activated DNA as a substrate Fig.

As expected, DNA polymerase activity was co-immunoprecipitated with PARP. In control experiments DNA polymerase activity was not associated either with the pre-immune serum or with protein A-Sepharose beads.

Furthermore, both enzymes were co-immunoprecipitated by specific antibodies to either PARP or DNA polymerase α data not shown from lysates of insect cells co-infected with recombinant baculovirus expressing both the p large subunit of DNA polymerase α 32 , 33 and PARP To exclude the possibility that this association could occur via tightly bound DNA fragments rather than by specific protein-protein interactions the immune complex was assayed for DNA polymerase activity in the absence or presence of DNase I-activated DNA as substrate.

Under the same experimental conditions PARP was found associated with DNA polymerase β only, in keeping with its potential role in base excision repair BER PARP interacts with DNA polymerase α-primase. A HeLa cell extracts were subjected to immunoprecipitation with anti-PARP lane 1 or two different anti-DNA polymerase α-primase antibodies SJK, lane 2; SJK, lane 3.

An anti-β-galactosidase antibody was used as a negative control lane 4. Immunoprecipitates were then analysed by immunoblotting using an anti-PARP antibody top.

Immunoprecipitates were also analysed by activity blot bottom. Figure 2 shows typical PARP and DNA polymerase patterns of doubly stained nuclei observed in proliferating HeLa cells.

Panels A and B show confocal images of DNA polymerase α-primase and PARP labelling respectively within the same nucleus. Co-localization yellow is observed at the nuclear periphery and in nucleoli; both patterns overlapped within the limits of the procedure.

Altogether, these results indicate that in vivo the two proteins are in close vicinity and are both preferentially present in the nuclear envelope. Confocal analysis of PARP and DNA polymerase α double staining in HeLa cells.

A Rhodamine-labelled DNA polymerase α red ; B fluorescein-labelled PARP green ; C merged image regions of overlap are in yellow. The interacting domains between PARP and DNA polymerase α-primase were mapped using two independent approaches. First, equimolar amounts of purified homogeneous human DNA polymerase α-primase and full-length PARP or PARP functional domains 29 kDa DBD or 40 kDa catalytic domain; Fig.

Then an anti-PARP antibody or the pre-immune serum was added to the reaction mixture and the immunoprecipitates were assayed for DNA polymerase α activity. As shown in Figure 3B , DNA polymerase activity co-immunoprecipitated with full-length PARP as well as with the 29 kDa DBD, but not with the 40 kDa catalytic domain or the pre-immune serum.

To determine whether this association also exists in vivo , in a second approach HeLa H cells constitutively expressing the human PARP 29 kDa domain as well as the parental cell line HpECV were used The proteins were identified by Western blot analysis using monoclonal anti-PARP antibody C1,9 17; Fig.

Lane 4 shows the typical pattern of cell line H expressing the recombinant 29 kDa DBD and also containing full-length endogenous PARP. Both proteins were immunoprecipitated with anti-PARP antibody lane 3 and with SJK lane 2 , but not with a non-specific anti-β-galactosidase antibody lane 1.

PARP was immunoprecipitated with SJK in HeLa cells, as already reported in Figure 1A. Thus both in vitro and in vivo PARP contacts DNA polymerase α-primase through its 29 kDa DBD.

In crude lysates obtained from the parental line HpECV no protein migrating at a molecular weight of 29 kDa was co-immunoprecipitated Fig. To assess the integrity of the PARP zinc fingers in this interaction 35 S-radiolabelled PARP functional domains were synthesized in vitro and used in Far western blot analysis.

Purified human DNA polymerase α-primase tetramer was separated by SDS-PAGE, transferred to nitrocellulose membrane and, after renaturation of the proteins, hybridized. As shown in Figure 3D , the kDa catalytic subunit of DNA polymerase α-primase bound to the full-length PARP as well as the 46 kDa DBD, whereas the 40 kDa C-terminal catalytic domain did not interact under these conditions.

Interestingly, the two mutated forms of the 46 kDa DBD failed to interact with the kDa catalytic subunit of DNA polymerase α-primase, strongly suggesting that integrity of the second zinc finger, at least, is required. From this Far western blot experiment we also concluded that the interaction is not mediated by DNA.

p and PARP were present throughout the cell cycle Fig. Cell lysates from different stages of the cell cycle were immunoprecipitated with the anti-DNA polymerase α-primase antibody SJK The immune complex was immunoblotted with both monoclonal antibody directed against the kDa subunit of DNA polymerase α-primase and anti-PARP antibody Fig.

Although DNA polymerase α-primase was efficiently immunoprecipitated throughout the cell cycle, PARP was found associated with DNA polymerase α-primase only during the S and G2 phases of the cell cycle, and not during G1 phase. The same amount of the large subunit p of DNA polymerase α-primase was present in both extracts.

The DNA binding domain of PARP interacts with the catalytic subunit of DNA polymerase α-primase. A Modular organization of the human PARP molecule.

B Purified DNA polymerase α-primase was incubated with PARP kDa or the purified domains of PARP 29 and 40 kDa. Immunoprecipitation was then performed with anti-PARP and the precipitates were assayed for DNA polymerase α activity at 0 and 30 min.

Immunoprecipitation with the pre-immune serum was performed as a negative control. C H cell lysates were immunoprecipitated with an anti-β-galactosidase antibody as a negative control lane 1 , anti-DNA polymerase α-primase antibody SJK lane 2 or anti-PARP antibody as positive control lane 3.

Aliquots of 10 µg H crude lysate were loaded as controls lane 4. HpECV cell lysates were immunoprecipitated with anti-DNA polymerase α-primase SJK lane 5.