Information

1.4: DNA Modifying Enzymes - Biology

1.4: DNA Modifying Enzymes - Biology


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Methylases

Just as the study of the bacterial restriction-modification system has provided a variety of specific endonucleases, there are also available a variety of specific DNA methylases.

  • The recognition sequences of the methylases are the same as the associated endonucleases (e.g. EcoR1 methylase recognizes and methylates at the sequence "GAATTC").
  • All methylases transfer the methyl group from S-adenosylmethionine (SAM) to a specific base in the recognition sequence, and SAM is a required component in the methylation reaction.
  • Methylation of DNA usually has the effect of protecting the DNA from the related restriction endonuclease. However, there are methylases with minimal specificity. For example, Sss I methylase will methylate cytosine residues in the sequence 5' … CG … 3'. In this case, the methylated DNA will be protected from a wide variety of restriction endonucleases.
  • Some restriction endonucleases will only cut DNA at their recognition sites if the DNA is methylated (e.g. Dpn I).
  • Still other restriction endonucleases will cut both methylated and non-methylated DNA at their recognition sequences (e.g. BamH I).

Dam and dcm Methylation

  • The methylase encoded by the dam gene (dam methylase) transfers a methyl group from SAM to the N6 position of the adenine base in the sequence 5' … GATC … 3'.
  • The methylase encoded by the dcm gene (dcm methylase) methylates the internal cytosine base, at the C5 position, in the sequences 5' … CCAGG … 3' and 5' … CCTGG … 3'.
  • Almost all strains of E. coli commonly used in cloning have a dam+dcm+ genotype. The point here is not that we particularly want our DNA to be methylated, but that to make a dam-dcm- host someone has to mutate the bacteria and isolate the correct mutant. That apparently has not been done for a lot of bacterial strains. Probably because the dam and dcm methylation affects only a small subset of potential restriction endonucleases

DNA isolated from dam+dcm+ strains will not actually be cut by a modest subset of available restriction endonucleases:

Recognition sequence
Restriction enzyme
GATC
GmeATC
TGATCA
Bcl I
+
-
GATC
Mbo I
+
-
ATCGAT
Cla I
+
-
TCTAGA
Xba I
+
-
TCGA
Taq I
+
-
GAAGA
Mbo II
+
-
GGTGA
Hph I
+
-

DNA may have to be prepared from E. coli strains which are dam-dcm- in order to be cut by these enzymes.

DNA Polymerases

A wide variety of polymerases have been characterized and are commercially available. All DNA polymerases share two general characteristics:

  1. They add nucleotides to the 3'-OH end of a primer
  2. The order of the nucleotides in the nascent polynucleotide is template directed

Figure 1.4.1: DNA Replication

In addition to the 5'->3' polymerase activity, polymerases can contain exonuclease activity. This exonuclease activity can proceed either in the 5'->3'direction, or in the 3'->5' direction.

  • Exonuclease activity in the 3'->5' direction allows the polymerase to correct a mistake if it incorporates an incorrect nucleotide (so called "error correction activity"). It can also slowly degrade the 3' end of the primer.
  • Exonuclease activity in the 5'->3' direction will allow it to degrade any other hybridized primer it may encounter. Without 5'->3' exonuclease activity, obstructing primers may or may not be physically deplaced, depending on the polymerase being used.

Different polymerases have differing error rates of misincorporation, and different rates of polymerization.

E. coli DNA polymerase I
E. coli DNA polymerase I - Klenow Fragment
T4 DNA polymerase
T7 DNA polymerase
Taq DNA polymerase
M-MuLV Reverse Transcriptase
5'->3' exonuclease activity
*
*
3'->5' exonuclease activity
*
*
*
*
Error Rate (x10-6)
9
40
<1
15
285
Strand Displacement
*
Heat Inactivation
*
*
*
*

Uses of polymerases

The various activities of the different polymerases lend them to a variety of applications. For example, restriction endonucleases can yield fragments of DNA with either 3' or 5' nucleotide "overhangs".

  • In the case of 5' overhangs, the 5'->3' polymerase activity can fill these in to make blunt ends.
  • In the case of 3' overhangs, the 3'->5' exonuclease activity present in some polymerases (especially T4 DNA polymerase) can "chew back" these ends to also make blunt-ended DNA fragments.

Figure 1.4.2: Polymerase activity

"Nick-translation"

This method is used to obtain highly radiolabeled single strand DNA fragments, which makes use of 5'->3' exonuclease activity present in some polymerases (E. coli DNA polymerase I, for example).

  • In this method a DNA duplex of interest is "nicked" (i.e. one of the strands is cut; see DNAse I).
  • Then DNA pol I is added along with radiolabeled nucleotides. The 5'->3' exonuclease activity chews away the 5' end at the "nick" site and the polymerase activity incorporates the radiolabeled nucleotides. The resulting polynucleotide will be highly radiolabeled and will hybridize to the DNA sequence of interest.

Figure 1.4.3: Nick-translation

  • Thermostable polymerases have the ability to remain functional at temperature ranges where the DNA duplex will actually "melt" and become separated. This has allowed the development of the "Polymerase Chain Reaction" technique (PCR), which has had a profound impact on modern Biotechnology. We will discuss this method at a later date.
  • The incorporation of dideoxy bases (i.e. no hydroxyl groups on either the 2' or 3' carbon of the ribose sugar) leads to termination of the polymerase reaction. This will be discussed in greater detail later. However, this chain termination by incorporation of dideoxynucleotides is the basis of the Sanger method of DNA sequencing, as well as therapies to try to inhibit viral replication.

Nucleases

Nuclease BAL-31

  • This is an exonuclease (starts at the termini and works inward) which will degrade both 3' and 5' termini of double-stranded DNA. It will not make internal cleavages ("nicks"), however, it will degrade the ends of DNA at existing internal "nicks" (which create both 3' and 5' termini).
  • The degradation of termini is not coordinated, meaning that the product is not 100% blunt-ended (even though the original duplex may have been blunt ended).
  • Such "ragged" ends can be made blunt by filling in and chewing back by a suitable polymerase (e.g. T4 DNA polymerase). The unit definition of 1 unit is the amount of enzyme required to remove 200 base pairs from each end of duplex DNA in 10 minutes at 30 °C.

Figure 1.4.4: Nuclease BAL-31 activity

Exonuclease III

  • Catalyzes the stepwise removal of nucleotides from the 3' hydroxyl termini of duplex DNA.
  • The enzyme will attack the 3' hydroxyl at duplex DNA with blunt ends, with 5' overhangs, or with internal "nicks".
  • Since duplex DNA is required, the enzyme will not digest the 3' end of duplex DNA where the termini are 3' overhangs.

Figure 1.4.5: Exonuclease III Activity

Mung Bean Nuclease (isolated from mung bean sprouts)

  • A single strand specific DNA and RNA endonuclease which will degrade single strand extensions from the ends of DNA and RNA leaving blunt ends.
  • The single strand extensions can be either 5' or 3' extensions - both are removed and a blunt duplex is left.

Figure 1.4.6: Mung Bean Nuclease activity

Deoxyribonuclease I (DNAse I) from Bovine pancrease

  • This enzyme hydrolyzes duplex or single DNA strands preferentially at the phosphodiester bonds 5' to pyrimidine nucleotides
  • In the presence of Mg2+ ion, DNAse I attacks each strand independently and produces nicks in a random fashion (useful for nick-translation)
  • In the presence of Mn2+ ion DNAse I cleaves both strands of DNA at approximately the same position (but leaving "ragged" ends)

Ligases

  • Ligases catalyze the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini of nucleotides (potentially RNA or DNA depending on the ligase).
  • In a sense, they are the opposite of restriction endonucleases, but they do not appear to be influenced by the local sequence, per se.
  • Ligases require either rATP or NAD+ as a cofactor, and this contrasts with restriction endonucleases.

The following are different types of ligases and their characteristics.

T4 DNA ligase

  • Isolated from bacteriophage T4.
  • Will ligate the ends of duplex DNA or RNA.
  • This enzyme will join blunt-end termini as well as ends with cohesive (complementary) overhanging ends (either 3' or 5' complementary overhangs).
  • This enzyme will also repair single stranded nicks in duplex DNA, RNA or DNA/RNA duplexes. Requires ATP as a cofactor.

Taq DNA ligase

  • This ligase will catalyze a phosphodiester bond between two adjacent oligonucleotides which are hybridized to a complementary DNA strand:

Figure 1.4.7: Taq DNA ligase activity

  • The ligation is efficient only if the oligonucleotides hybridize perfectly with the template strand.
  • The enzyme is active at relatively high temperatures (45 - 65 °C). Requires NAD+ as a cofactor.

T4 RNA ligase

  • Will catalyze formation of a phosphodiester bond between RNA/RNA oligonucleotides, RNA/DNA oligonucleotides, or DNA/DNA oligonucleotides.
  • Requires ATP as a cofactor.
  • This enzyme does not require a template strand.

T4 RNA ligase can be used for a variety of purposes including constructing RNA/DNA hybrid molecules.

Figure 1.4.8: T4 RNA ligase activity

DNA ligase (E. coli)

  • Will catalyze a phosphodiester bond between duplex DNA containing cohesive ends.
  • It will not efficiently ligate blund ended fragments.
  • Requires NAD+ as a cofactor.

Figure 1.4.9: DNA ligase (E. Coli) activity

T4 polynucleotide kinase

  • Catalyzes the transfer and exchange of a phosphate group from the g position of rATP (adenine ribose triphosphate nucleotide) to the 5' hydroxyl terminus of double stranded and single stranded DNA or RNA, and nucleoside 3' monophosphates.
  • The enzyme will also remove 3' phosphoryl groups.
  • Oligonucleotides which are obtained from automated synthesizers lack a 5' phosphate group, and thus, cannot be ligated to other polynucleotides.

T4 polynucleotide kinase can be used to phosphorylate the 5' end of such polynucleotides:

Figure 1.4.10: T4 polynucleotide kinase activity

Calf intestinal phosphatase (CIP)

  • Catalyzes the removal of 5' phosphate groups from RNA, DNA and ribo- and deoxyribo- nucleoside triphosphates (e.g. ATP, rATP).
  • CIP treated duplex DNA cannot self ligate.
  • Hemi-phosphorylated duplexes will be ligated on one strand (the phosphorylated strand) and remain "nicked" on the other.

Figure 1.4.11: CIP activity


T4 DNA Ligase ( MB007 )

Description: T4 DNA Ligase is an ultrapure recombinant enzyme purified from Escherichia coli supplied with an optimized 10× Reaction Buffer, which includes ATP. T4 DNA Ligase catalyses the formation of a phosphodiester bond between juxtaposed 5′-phosphoryl and 3′-hydroxyl termini in duplex DNA. It repairs single-strand nicks in duplex DNA and will join both blunt and cohesive-end restriction fragments of duplex DNA or RNA. The enzyme requires ATP as cofactor.

Features:
– Free from detectable nonspecific nuclease, endonuclease, RNase and DNase activities
– Supplied with an optimized reaction buffer for efficient ligation reactions

Applications:
– Cloning of both blunt- and sticky (cohesive)-end restriction fragments
– Cloning of PCR products
– Joining linkers and adapters to DNA
– Nick repair
– Self-circularization of linear DNA

Specifications:

Enzyme concentration: 5 U/μL
Source: Recombinant E. coli strain
Optimal reaction temperature: 16-20 °C
Heat inactivation: 65 °C for 10 min
Storage conditions: Store at -20 °C
Shipping conditions: Shipped at dry ice

Components:
– T4 DNA Ligase (5 U/μL)
– Reaction buffer (4x)


Molecular Cell Biology. 4th edition.

DNA molecules can coil and bend in space, leading to changes in topology, including formation of negative or positive supercoils. For example, as discussed in Chapter 4, local unwinding of a DNA duplex whose ends are fixed causes stress that is relieved by supercoiling. The enzymes that control the topology of DNA function at several different steps in replication in both prokaryotic and eukaryotic cells. In this section we describe the two different classes of topoisomerases and their role in DNA replication.


DNase I (RNase free)

Applications:
DNase I is commonly added to cell lysis reagents to remove the viscosity caused by the DNA content in bacterial cell lysates or to remove DNA templates from RNA produced by in vitro transcription.
DNase I removes unwanted DNA from cell lysates to improve protein extraction efficiency.

Description:
DNase I (RNase free), Deoxyribonuclease I is a single, glycosylated polypeptide that degrades single- and double-stranded DNA. The enzyme works by cleaving DNA into 5' phosphodinucleotide and small oligonucleotide fragments.
DNase I is used for application requiring the digestion of DNA in which it is crucial to avoid damage to RNA.

Content:
DNase I (RNase free)
2 units/&mul DNase I in 10 mM Tris-HCl pH 7.5, 10 mM CaCl2, 10 mM MgCl2 and 50 % [v/v] glycerol

DNase I Reaction Buffer
10 x conc. reaction buffer containing 100 mM Tris-HCl pH 7.6 (25°C), 25 mM MgCl2, 5 mM CaCl2

Reaction Conditions
1x DNase I Reaction Buffer
Incubation at 37°C
High levels of monovalent ions such as Na + and K + (i.e. 100 mM) may decrease DNase I activity.

Inactivation:
DNase I is completely inactivated by incubation at 65 °C for 10 minutes.

Activity:
> 2500 units/mg protein


[1] DNase I activity is also measured in 'Degradation Assay units' defined as the amount of enzyme required to completely degrade 1 &mug of plasmid DNA in 10 minutes at 37 °C in 10 mM Tris-HCl pH 7.5, 50 mM MgCl2 and 13 mM CaCl2.
1 ‘Degradation Assay unit’ is equivalent to 0.3 ‘Kunitz units’.

Product Citations:
Please click the black arrow on the right to expand the citation list. Click publication title for the full text.

Selected References:
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press 10.6
Tabor et al. (1997) DNA-Dependent DNA Polymerases. In: Current Protocols in Molecular Biology. Ausubel et al., eds. Wiley & Sons Inc. 3.5.4-6.
Pan et al. (1999) Ca2+- dependent activity of human DNase I and its hyperactive variants. Protein Sci. 8:1780.


DNA Polymerase Selection Chart

DNA Ligases form phosphodiester bonds between DNA strands, joining them together. DNA Ligase is responsible for repairing breaks in single and double stranded DNA. In molecular biology labs, DNA Ligase is commonly used for molecular cloning applications.

Each DNA Ligase has special characteristics, making it useful for specific applications. Knowing these characteristics enables you to pick the right ultra pure DNA Ligase for your application.


Americas

Site will be displayed in English.

We use these cookies to ensure our site functions securely and properly they are necessary for our services to function and cannot be switched off in our systems. They are usually only set in response to actions made by you which amount to a request for services, such as logging in, using a shopping cart or filling in forms. You can set your browser to block or alert you about these cookies, but some parts of our services will not work without them. Like the other cookies we use, strictly necessary cookies may be either first-party cookies or third - party cookies.

We use these cookies to remember your settings and preferences. For example, we may use these cookies to remember your language preferences.
Allow Preference Cookies

We use these cookies to collect information about how you interact with our services and to help us measure and improve them. For example, we may use these cookies to determine if you have interacted with a certain page.
Allow Performance/Statistics Cookies

We and our advertising partners use these cookies to deliver advertisements, to make them more relevant and meaningful to you, and to track the efficiency of our advertising campaigns, both on our services and on other websites and social media.
Allow Marketing Cookies


T4 DNA Ligase

Unit Definition: One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmol of 32 P from pyrophosphate to ATP, into Norit-adsorbable material in 20 minutes at 37 °C.

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Form: liquid (Supplied in 10 mM Tris-HCl pH 7.4, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 &mug/ml BSA and 50 % [v/v] glycerol)

Concentration: 2.5 Weiss units/&mul (500 CE units/&mul)

Description:
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3'-hydroxyl termini in duplex DNA or RNA.

Content:
Standard Ligation Buffer, 10x conc.
500 mM Tris-HCl pH 7.8 at 25 °C, 100 mM MgCl2, 100 mM DTT, 10 mM ATP and 25 &mug/ml BSA

Fast Ligation Buffer, 2x conc.
60 mM Tris-HCl pH 7.8 at 25 °C, 20 mM MgCl2, 20 mM DTT, 2 mM ATP and 10 % PEG

componentEN-149SEN-149L
T4 DNA Ligase160 &mul5 x 160 &mul
Standard Ligation Buffer, 10x conc.1 ml5 x 1 ml
Fast Ligation Buffer, 2x conc.5 ml5 x 5 ml

Heat inactivation:
T4 DNA Ligase can be inactivated by incubation at 65 °C for 10 minutes.

  • One Cohesive-End Ligation Unit (CEU) is defined as the amount of enzyme required to give 50 % ligation of Hind III fragments of λ DNA (5' DNA termini concentration of 0.12 &muM, 300 &mug/ml) in a total reaction volume of 20 &mul in 30 minutes at 16 °C in 1x T4 DNA Ligase Reaction Buffer.
  • One Weiss unit is equivalent to approx. 200 CE units.
  • T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200 mM.
  • Ligation of blunt-ended and single-base pair overhang fragments requires about 50 times as much enzyme to achieve the same extent of ligation as cohesive-end DNA fragments. Blunt-end ligation may be enhanced by addition of PEG 4000 (10 % w/v final concentration) or hexamine chloride, or by reducing the ATP concentration to 50 &muM.
  • To dilute T4 DNA Ligase for subsequent storage at -20 °C a storage buffer containing 50 % glycerol should be used, to dilute Ligase for immediate use, 1x Reaction Buffer is recommended.


Assay Set-Up:
Standard Ligation Assay

comp.final amount/conc.20 &mul assay
Standard Ligation Buffer, 10x conc.1x2 &mul
Vector/Insert DNA100 ng - 1 &mug100 ng - 1 &mug
T4 DNA Ligase0.1 - 1 Weiss units0.04-0.4 &mul
PCR-grade Water-fill up to 20 &mul

Incubate for 20 - 30 min at 16 °C for optimal ligation.

comp.final amount/conc.20 &mul assay
Fast Ligation Buffer, 2x conc.1x10 &mul
Vector/Insert DNA100 ng - 1 &mug100 ng - 1 &mug
T4 DNA Ligase0.1 - 1 Weiss units0.04-0.4 &mul
PCR-grade Water-fill up to 20 &mul

Incubate for 5 min for cohesive-ended ligations or 15 min for blunt-ended ligations at ambient temperature (20 - 25 °C).

Product Citations:
Please click the black arrow on the right to expand the citation list. Click publication title for the full text.


Restriction Enzyme Digestion

Preparation of DNA for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together. The DNA to be cloned can vary widely, from genomic DNA extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved from one vector to another (subcloning). Restriction enzymes can also be used to generate compatible ends on PCR products. In all cases, one or more restriction enzymes are used to digest the DNA resulting in either non-directional or directional insertion into the compatible plasmid.

Genomic DNA, regardless of the source, is typically digested with restriction enzymes that recognize 6-8 consecutive bases, as these recognition sites occur less frequently in the genome than 4-base sites, and result in larger DNA fragments. The desired insert size for the clone library determines which enzymes are selected, as well as the digestion conditions. Most often, a serial dilution of the selected restriction enzyme(s) is used to digest the starting material and the desired insert size range is isolated by electrophoresis followed by gel extraction of the DNA. This method of preparation provides DNA fragments of the desired size with ends compatible to the selected vector DNA.

Subcloning requires the use of 1-2 restriction enzymes that cut immediately outside the insert fragment without cutting within the insert itself. Restriction enzymes that have a recognition site within the multiple cloning site (MCS) are commonly used since they do not cut elsewhere in the vector DNA and typically produce two easily resolved DNA fragments. The gene of interest is most commonly subcloned into an expression vector for improved protein expression and/or addition of a purification tag. In this case, it is essential that the gene be inserted in the correct orientation and in frame with the transcription promoter.

The Polymerase Chain Reaction (PCR) is commonly used to amplify a gene or DNA fragment of interest, from any source of DNA, to be cloned. In order to generate compatible ends, it is common to add restriction sites to the 5&rsquo end of both PCR primers. When adding restriction sites to a PCR primer, it is recommended to include 6 bases between the recognition site and the 5&rsquo end of the primer. These additional bases provide sufficient DNA for the restriction enzyme to bind the recognition site and cut efficiently. When selecting a restriction site(s) to add to the primers, it is important to determine which site(s) will be compatible with your selected vector, whether directional cloning is desired and, most importantly, confirm that the recognition site(s) does not occur within the gene or DNA fragment.


Enzymes involved in DNA Replication

This is the list of Enzymes Involved in DNA Replication. Let us discuss this in detail…

  • Single-Stranded Binding Protein (SSBP)
  • DNA Helicases
  • Topoisomerases
  • DNA primase
  • DNA Ligase
  • DNA polymerases

1. Single-Stranded Binding Protein (SSBP)

SSBP means Single-Stranded Binding Proteins. It has a very important role in DNA Replication in E.Coli.

Single-stranded binding proteins bind to and stabilize single-stranded DNA during DNA replication until the single-stranded DNA can be used as a template for a new strand to bind to.

The SSB proteins attached with both the lagging strand and the leading strand to prevent re-association of the strands.

2.DNA Helicase

  • DNA helicase enzyme functions “Unwinds DNA”.
  • They have molecular weight 300,000, which contains SIX identical subunits.
  • Okazaki fragments” are short stretches of 1000-2000 bases produced during discontinuous replication, they are later joined into a covalently intact strand.
  • The “Dna.B helicase” and “Dna.G Primase” constitute a functional unit within the replication complex, called the “PRIMOSOME”.
  • The DNA is around by the Dna.B helicase at the replication fork, DNA primase occasionally associates with Dna.B helicase and synthesizes a short RNA primer.
  • ”Helicase” and “Nuclease” activities of the Rec B, C, D enzyme is believed to help initiate homologous genetic recombination in E.Coli. It is also involved in the repair of double-strand breaks at the collapsed replication fork.
  • A“Helicase” is an enzyme that separates the strands of DNA usually the hydrolysis of ATP to provide the necessary energy.

3. Topoisomerases

  • Topoisomerase is also known as “DNA Gyrase”.
  • “Topoisomerases” is an enzyme that can change the “Linking number”(Lk).
  • Every cell has enzymes that increase (or) decrease the extent of DNA unwinding is called “Topoisomerases” the property of DNA that they change is the linking number.
  • Topoisomerases”, these enzymes play an especially important role in processes such as “Replication” and “DNA packaging”.
  • There are two classes of topoisomerases.
    • Type-I Topoisomerases
    • Type-II Topoisomerases

    A) Type-I Topoisomerases:

    This act by transiently breaking one of the two DNA strands, rotating one of the ends of the unbroken strand, and rejoining the broken ends they change Lk in increments of 1.

    B) Type-II Topoisomerases:

    The enzyme breaks both DNA strands and changes Lk in increments of 2.

    Prokaryotic Topoisomerases

    FOUR different Topoisomerases (I and IV) occur in E.Coli.

    1) Type.I (Topoisomerase I and III):

    The type I generally relax DNA by removing negative super-coils (increasing Lk)

    2) Type.II (Topoisomerase II and IV):

    The Topoisomerase II is also called “DNA gyrase”, can introduce negative supercoils. (Decrease Lk).

    • It uses the energy of ATP and a surprising mechanism to accomplish this.
    • The degree of supercoiling of bacterial DNA is maintained by regulation of the net activity of topoisomerase-I and II.

    Eukaryotic Topoisomerases

    Eukaryotic cells also have type-I and type-II topoisomerases. Topoisomerases-I & II are both type-I. The two type-II topoisomerases, topoisomerases IIa and IIb, can not unwind DNA (introduce negative supercoils).

    Although both can relax positive and negative supercoils. We consider one probable origin of negative supercoils in eukaryotic cells.

    • The DNA gyrase molecular weight is 400,000, which contains FOUR subunits and functions “Supercoiling”.
    • Supercoiled DNA is a higher-ordered structure occurring in circular DNA molecules wrapped around a core.

    What is the linking number?

    The linking number (Lk) is a topological property. Lk can be defined as “ the number of times the second strand pierces the second strand surface”.

    4. DNA Primase

    In replication, before DNA polymerase iii can begin synthesizing DNA primers must be present on the template generally short segments of RNA synthesized by an enzyme called “Primases”.

    • DNA primase has molecular weight 60,000 Dalton and contains only a single subunit, which functions synthesize RNA primers.
    • The Dna.B helicase and Dna.G primase constitute a functional unit within the replication complex, called the “Primosome”.
    • The RNA primer typically is 15-50 bases long. It synthesizes primers starting with the sequence pppAG, opposite the sequence 3’-GTC-5’ in the template.

    5. DNA Ligase

    An enzyme that creates a phosphodiester bond between the 3’ end of one DNA segment and the 5’ end of another. Once the RNA primer has been removed and replaced the adjacent Okazaki fragments must be linked together. The 3’-OH end of one fragment is adjacent to the 5’-Phosphate end of the previous fragment. The responsible for sealing this nick lies with the enzyme DNA ligase. Ligases are present in both prokaryotes and eukaryotes.

    Mechanism of Enzyme activity:

    The E.Coli and T4 ligases share the property of sealing nicks that have 3’’-OH and 5’- P termini. Both enzymes undertake a two-step reaction, involving an ‘enzyme-AMP complex’.

    The AMP of the enzyme complex becomes attached to the 5’-Phosphate of the nick and then a phosphodiester bond is formed with the 3’-OH terminus of the nick, releasing the enzyme and the AMP.


    Author information

    Affiliations

    Division of Signaling and Gene Expression, La Jolla Institute for Immunology, 9420 Athena Circle, La Jolla, CA, 92037, USA

    Atsushi Onodera, Edahí González-Avalos, Chan-Wang Jerry Lio, Romain O. Georges & Anjana Rao

    Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Atsushi Onodera & Toshinori Nakayama

    Institute for Global Prominent Research, Chiba University, 1-33, Yayoicho, Inage-ku, Chiba, 263-8522, Japan

    Bioinformatics and Systems Biology Graduate Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA

    Present address: Department of Microbial Infection and Immunity, Ohio State University, 460 W 12th Ave, Columbus, OH, 43210, USA

    Cancer Signaling and Epigenetics Program & Cancer Epigenetics Institute, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA

    AMED-CREST, AMED, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Department of Pharmacology and Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA

    Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA, 92037, USA

    You can also search for this author in PubMed Google Scholar

    You can also search for this author in PubMed Google Scholar

    You can also search for this author in PubMed Google Scholar

    You can also search for this author in PubMed Google Scholar

    You can also search for this author in PubMed Google Scholar

    You can also search for this author in PubMed Google Scholar

    You can also search for this author in PubMed Google Scholar

    Contributions

    A.O. acquired, analyzed, and interpreted the data. E.G.-A. performed some of the bioinformatic analyses. C.-W.L. provided CMS-IP datasets in BMDMs. R.O.G. received and maintained the Tdg fl/fl mice and designed and carried out the breeding for conditional Tdg deletion in mice. A.B. provided the Tdg fl/fl mice and advice on some experiments. T.N. provided advice and key reagents for the T-cell experiments. A.O. and A.R. conceptualized the experiments, interpreted the data, and wrote the manuscript. All authors were involved in reviewing and editing the manuscript. The authors read and approved the final manuscript.

    Corresponding author


    Watch the video: DNA Modifying Enzymes. Recombinant DNA Technology. GATE BT. Gurmantra (February 2023).