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1 DNA Structure and Replication

Apr 08, 2015

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Page 1: 1 DNA Structure and Replication

DNA Structure and Replication

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  DNA = deoxyribonucleic acid   - polymer of deoxyribonucleotides   base + deoxyribose + phosphate

  RNA = ribonucleic acid   - polymer of ribonucleotides   base + ribose + phosphate

Nucleotide = base + sugar + phosphate

Types of Nucleic Acids:

Nucleic Acids – polymers of nucleotides Primary structure is the sequence of nucleotides attached to one another by

phosphodiester bonds

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Different kinds of phosphate bonds

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Nitrogenated bases are purines and pyrimidines

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Cytosine can be converted to uracil by cytosine deaminase

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  Composed of monodeoxyribonucleotides covalently linked by 3’ 5’ phosphodiester bonds

  Has polarity

  Bases always written in sequence from 5’end of chain on the left to the 3’ end on the right

DNA – a polydeoxyribonucleotide

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Complementarity of the base pairs enable stacking in the energetically most favorable condition in the double helix

The main glue for the double helix is the stacking of bases between adjacent bases

 The stacking of adjacent bases stabilize single stranded DNA and double stranded DNA

 In double stranded DNA the separation between the hydrophobic bases and the hydrophilic components (sugar and phosphate) is more pronounced than the single stranded DNA thereby making more energetically favorable

 Additional stability comes from hydrogen bonds between the bases. Indirectly they optimize, because of their dependence in directionality, the stacking of bases

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Chargaff’s rules of base composition

 The total amount of pyrimidine nucleotides (T +C) always equals the total amount of purine nucleotides (A + G)

  The amount of T always equals the amount of A, and the amount of C always equals the amount of G

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  In any double-stranded DNA, A = T and G = C. Purines = pyrimidines

 Given the sequence of bases on 1 chain, the sequence of bases on the complementary chain can be determined

  Tautomers of the bases give rise to configurational isomers whereby the hydrogen location differs

One polynucleotide chain of the DNA double helix is always the complement of the other

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Double-stranded DNA exists in 3 forms (A, B and Z) B form is usually found under

physiologic conditions DNA

RNA

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Comparisons of DNA double-stranded forms

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  Denaturation (Melting) of DNA   Separation of the 2 DNA strands in the double helix   Occurs when H-bonds between the paired bases are disrupted   Occurs in the laboratory if solution is heated   The higher the GC content, the higher the Tm, the temperature at

which ½ of the helical structure is lost

  Renaturation (Reannealing)   Process by which complementary DNA strands reform the double helix

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Packaging of DNA Inside the Cell

  dsDNA

  Histones

  Nucleosomes

  Linker DNA

  Nucleofilament

  30 nm fiber

  Protein Scaffold

  Chromosome

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Because DNA molecules are so large, they require special packaging to enable them to reside within cells

 E. coli: DNA has 4 x 106 base pairs, 2 mm long

  Circular DNA is supercoiled

 Human: DNA contains a total of 4 x 109 base pairs

  DNA from all 46 chromosomes in a diploid human cell, placed end to end, would stretch about 2 m (> 6 ft)

  DNA has multiple levels of packaging that is facilitated by binding with histones

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Intermediate filaments are like ropes made of long, twisted strands of protein like the nuclear lamins

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Nuclear lamins are fibrous proteins that form intermediates filaments

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  Positively charged at physiologic pH  Result of high content of lysine

and arginine  Form ionic bonds with

negatively charged DNA  Help neutralize the negatively

charged DNA phosphate groups  They contain an N-terminal tail

and a globular histone fold (at least three alpha helices connected by short loops)

  With 5 classes: H1 – associated with linker DNA H2A H2B H3 H4

Form the core of a nucleosome

Histones – small proteins

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DNA Packaging   DNA double helix are wound nearly twice

around a core of histones (2 each of H2A, H2B, H3 and H4)=

nucleosome

  The histone H3-H4 dimer and H2A-H2B dimer are formed from the handshake interaction

  An H3-H4 tetramer forms and binds to the DNA

  Two H2A-H2B dimers are then added, to complete the nucleosome

  Note that all eight N-terminal tails of the histones protrude from the disc-shape core structure

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Nucleosomes are joined by “linker” DNA (Nucleofilaments)

Linker DNA: about 50 base pairs long associated with H1

  corresponds to an extended polynucleosome chain   has the “beads-on-a-string” appearance

  H1 – tissue-specific and species-specific of histones facilitates the packing of nucleosomes into more compact structures

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Chromatin packing represents a compaction of 10,000 fold

Chromosome structure found in interphase

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  Semi-conservative  half of the parental DNA molecule (1 strand)

 conserved in each new double helix

  paired with a newly synthesized complementary strand

  Exhibits polarity

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The DNA replication fork is asymmetrical  DNA polymerases

–  Responsible for copying the DNA templates –  Synthesize the new DNA strands in the 5’ 3’ direction –  The parental nucleotide strand is read in the 3’ 5’ direction

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Replication in prokaryotes   Separation of the 2 complementary DNA strands (helicase)

  Single-stranded DNA binding proteins   Topoisomerases

  Primer synthesis (an RNA primer required)

  DNA synthesis   DNA polymerase III (prokaryotes)   Okazaki fragments on lagging strand   Primers removed by DNA polymerase I, which fills in gaps   DNA Ligase

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 DNA helicases   move into neighboring double-stranded region, forcing the strands apart, in effect unwinding the helix

 When strands separate, SSB proteins bind, preventing reformation of the double helix

 Single-stranded DNA-binding (SSB) proteins (helix destabilizing proteins) bind to single-stranded DNA

 SSB proteins   keep the 2 strands of DNA separated in the area of the

replication origin   also protect the DNA from nucleases that cleave

single-stranded DNA

Replication in Prokaryotes Special proteins help to open up the DNA double helix in front of the

replication fork

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Primer synthesis Replication in Prokaryotes

Separation of the 2 Complementary DNA strands

 Primase – DNA-dependent RNA polymerase  Synthesizes short stretches of RNA (about 10 nucleotides

long) that are complementary and anti-parallel to the DNA template

 DNA polymerases require an RNA primer with a free hydroxyl group on the 3’-end of the RNA strand   Hydroxyl group serves as the first acceptor of a nucleotide by action of DNA polymerase

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DNA synthesis Replication in Prokaryotes Separation of the 2 Complementary DNA strands Primer Synthesis

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  DNA Polymerase III  The chemistry of DNA replication, repair and recombination is largely that of the phosphodiester bonds that link neighboring nucleotides in a DNA chain

  Has 5’ 3’ polymerase activity   Uses 5’-deoxyribonucleotide triphosphates

  All 4 deoxyribonucleotide triphosphates must be present (dATP, dTTP, dCTP, dGTP)

  DNA synthesis stops if (when) a nucleotide is depleted

 The elongation of the 3OH end involves a transesterification reaction

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DNA synthesis Replication in Prokaryotes

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Initiation of DNA replication

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Replication in prokaryotes

  Separation of the 2 Complementary DNA strands   Leads to the formation of a “V” where active DNA synthesis occurs

  The replication fork moves along the DNA molecule as synthesis occurs using a polymerase dimer

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DNA synthesis on the leading and lagging strands

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The chemistry of DNA synthesis  The addition of a deoxyribnucleotide to the 3’ end of a polynucleotide chain (the primer strand) is the fundamental reaction by which DNA is synthesized

 Base-pairing between an incoming deoxyribonucleotide triphosphate and an existing strand of DNA (template strand) guides the formation of DNA and causes it to have a complementary nucleotide sequence

 The shape of DNA polymerase resembles a right hand in which the palm, fingers, and the thumb grasp the DNA and form the active site

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The high fidelity of DNA replication requires several proofreading mechanisms

 The fidelity of copying DNA during replication is such that only about 1 mistake occurs for every 109 nucleotides copied

 If the DNA polymerase did nothing special when a mispairing occurred between an incoming deoxyribonucleotides triphosphate and the DNA template, the wrong nucleotide would be incorporated in the DNA chain, producing frequent mutations

 The high fidelity of DNA replication, however, depends not only in the initial-base pairing but also on several “proofreading” mechanisms that act sequentially to correct any initial mispairing that might have occurred.

 DNA polymerase performs the first proofreading step just before a new nucleotide is added to the growing chain 35

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Only DNA replication in the 5’ to 3’ direction allows efficient error correction

 The need for accuracy probably explains why DNA replication occurs only in the 5’ to 3’ direction

 If there were a DNA polymerase that added deoxyribonucleoside triphophate in the 3’ to 5’ direction, the growing 5’-chain end, rather than the incoming mononucleotide, would provide the activating triphosphate needed for the covalent linkage

 In this case, the mistakes in polymerization could not be simply hydrolyzed away, because the bare 5’-chain end thus created would immediately terminate DNA synthesis

 It is therefore possible to correct mismatched base only if it has been added to the 3’ end of a DNA chain 36

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5’ 3’

5’ 3’

5’ 3’

5’ 3’

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3’ 5’

A G C T G C G G A T C!T

5’ Replication

DNA Polymerase III

  DNA Polymerase III

- has 5’ 3’ polymerase activity

 Adds deoxyribonucleotides one at a time to the 3’ end of the growing chain

 Sequence of nucleotides added is dictated by the base sequence of the template strand with which the incoming nucleotides are paired

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C!T

5’

 DNA Polymerase III  has 5’ 3’ polymerase activity

DNA synthesis

C!

Replication in Prokaryotes

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G C T C

3’ 5’

A G C T G C G G A T

5’

DNA polymerase III recognizes its mistake

  DNA Polymerase III  has proofreading activity

DNA synthesis Replication in Prokaryotes

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C T C

3’ 5’

A G C T G C G G A T

5’

  DNA Polymerase III has proofreading activity

DNA synthesis

DNA polymerase III uses its 3’ 5’ exonuclease activity to backup

Replication in Prokaryotes

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C T C

3’ 5’

A G C T G C G G A T

5’

  DNA Polymerase III - has proofreading activity

DNA synthesis

A

DNA polymerase III repairs the mismatch.

Replication in Prokaryotes

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C T C

3’ 5’

A G C T G C G G A T

5’

 DNA Polymerase III  has proofreading activity

DNA synthesis

A

DNA polymerase III continues with its 5’ 3’ activity.

G

Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C U C A

RNA Primer

Newly synthesized DNA

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C U C A A G G

Once DNA polymerase III reaches the RNA primer, it can proceed no further

  DNA Polymerase III

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C U C A A

 DNA Polymerase III

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C U C A A G

  DNA Polymerase III

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C U C A A G G

DNA polymerase III dissociates from the DNA strand.

DNA polymerase I takes its place

  DNA Polymerase I

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C U C A A G G

DNA polymerase I has:   5’ to 3’ exonuclease activity

 Able to remove the RNA primer

  5’ to 3’ polymerase activity  Replaces the removed RNA primer with deoxyribonucleotides

  3’ to 5’ exonuclease proofreading activity

  DNA Polymerase I

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C U C A A G G

  DNA Polymerase I

DNA synthesis

DNA polymerase I has:   3’ to 5’ exonuclease activity

  “proofreads” the newly synthesized DNA chain

Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C T C A A G G

  DNA Polymerase I

DNA synthesis

 removal/synthesis/proofreading continues one nucleotide at a time until the RNA is totally degraded and the gap is filled with DNA

Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C T C A A G G

C

U

Nick in the phosphodiester backbone

  DNA Polymerase I

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C T C A A G G

C

U

DNA ligase seals the nick

  DNA ligase

DNA synthesis Replication in Prokaryotes

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3’ 5’

A G C T G C G G A T C T

5’

C C T C A A G G

C

A

  DNA ligase

DNA synthesis Replication in Prokaryotes

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Summary of key components of DNA replication in prokaryotes on the lagging

strand

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DNA synthesis

  Catalyzes the final phosphodiester linkage between: a. 5’ phosphate group on the DNA chain synthesized by DNA polymerase III and

b. 3’ hydroxyl group on the chain made by DNA polymerase I

Replication in Prokaryotes DNA ligase

High E

H E Is kept

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Comparison of exonucleases and endonucleases

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DNA replication in eukaryotes occurs in the S phase

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DNA replication in eukaryotes (2)   Primase activity of polymerase α/primase

complex initiates synthesis of an RNA primer

  After RNA primer reaches 10 nucleotides, the polymerase α 5’ 3’ polymerase activity takes over   Adds about 20-30 deoxyribonucleotides

  Polymerase α/primase dissociates

  Polymerase δ complex binds and elongates the chain.

  As the replication complex approaches an earlier RNA primer, the primer is degraded by RNAase H and FEN 1

  Gap is filled by polymerse δ continued elongation of the Okazaki fragment

  Remaining nick is sealed by DNA ligase

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A) Small prokaryotic circular DNA contains only one origin of replication

B) Very long eukaryotic DNA contains from 400 to 10000 orings of replication

Replication of DNA:origins and replication forks A stretch of DNA that is rich in As and Ts and thereby easy to unwind

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Eukaryotic DNA replication   Similar to that in prokaryotes

  Daunorubicin and doxorubin are used as in the treatment of leukemias.

  They interfere with the activity of topoisomerase II by intercalating between bases of DNA. This prevent DNA replication in tumor cells

  Differences:  With multiple origins of replication   Centromeres (they allow separation of chromosomes into each daughter cell)   Linear DNA   Telomerase is required to maintain the length of the chromosome   RNA primers are removed by RNAse H   The primer is a mixture of RNA and DNA   RNAase H and Fen degrades the primer   Eukaryotic polymerases

•  DNA polymerase α and δ : lagging strand synthesis •  DNA polymerase δ : leading strand synthesis (α initiates) •  DNA polymerase γ: mitochondrial DNA synthesis •  DNA polymerase β and ε: DNA repair

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Types of DNA polymerases

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  DNA topoisomerases alter the linking number of DNA   With both nuclease (strand-cutting) and ligase (strand-resealing) activities   With 2 types:

  Type I DNA topoisomerase   Type II DNA topoisomerase

Replication in Prokaryotes

DNA topoisomerases add or remove supercoils in the helix

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L = T + W

L=linking number (defined as the number of times one strand of DNA winds around the other)

T= turns

W= writhing number refers to the number of super-helix turns

A

B

C

D

Topoisomers are DNA molecules differing only in linking number

Lk = Number of nucleotides 10.5

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Replication in Prokaryotes

DNA topoisomerases remove supercoils in the helix

Type I topoisomerase

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Topoisomerase I (heterotetramer of gyrA and gyr B) mechanism involves phosphotyrosine intermediates

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Replication in Prokaryotes

DNA topoisomerases remove or add supercoils in the helix

Type II topoisomerase

Viral DNA

5’ 30’ Topo II mins

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 A type II topoisomerase found in E. coli  Able to introduce negative supercoils into relaxed circular

DNA using energy from the hydrolysis of ATP  Facilitates the future replication of DNA because the (-) supercoils

neutralize the positive supercoils introduced during opening of the double helix

 A swivel ahead of the replication machinery  DNA gyrase is the target of many antibiotics.

 Topoisomerase inhibitors such as nalidixic acid freeze the covalent DNA-protein links; nalidixic acid is used for urinary tract infections. Ciprofloxacin is another inhibitor of topoisomerases which is used to prevent and treat antrax

Replication in Prokaryotes DNA gyrase

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Inhibitors of topoisomerases in eukaryotes and prokaryotes

Antibiotics

Anticancer drugs

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Reverse transcriptase

 Uses RNA as template for the synthesis of DNA

 Present in some viruses

  In contrast to DNA polymerase, RNA polymerase do not have 3’ 5 proofreading activity thereby is prone to errors and high rate of mutations

 Example: human immunodeficiency virus

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Production of the virus that causes AIDS

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Only eukaryotes contain genomes in which some genes have introns

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The replication problem at chromosome ends in linear chromosomes

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 Consist of many tandem repeats of a 6-nucleotide DNA sequence (TTGGGG tetrahymena  And human TTAGGG)

 Do not encode proteins

 Undergo cycles of shortening and addition of new repeats to the 3’ end by telomerase

 Telomerase is active in germ cells, fetal cells and cancer cells  Generally not active in adult somatic cell

 Generally not active in adult somatic cells

Telomeres

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Mechanism of action of telomerase

78 Telomerase has reverse transcriptase activity

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Central dogma of molecular biology

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List the forces that hold the DNA double helix together as a stable unit - Hydrogen bonding, dipole interaction

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What is the polarity of the growing leading strand synthesize by DNA polymerase III?

What is the polarity for the lagging strand?

What is the polarity of the template in both cases?

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If species A has more DNA per nucleus than species B, does A necessarily have more genes than B?

Please explain

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DNA topoisomerases play important roles in DNA replication and supercoiling. These enzymes are also the targets for certain anticancer drugs. Eric Nelson and his colleagues studied m-AMSA, one of the anticancer compounds that acts on topisomerase enzymes. They found that m-AMSA stabilizes an intermediate produced in the course of the topoisomerase’s action. The intermediate consisted of the topoisomerase bound to the broken ends of the DNA. Breaks in DNA that are produced by anticancer compounds such as m-AMSA inhibit the replication of the cellular DNA and thus stop cancer cells from proliferating.

Propose a mechanism for how m-AMSA and other anticancer agents that target topoisomerase enzymes taking part in replication might lead to DNA breaks and chromosome rearrangments.

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A closed-circular DNA molecule in its relaxed form has an Lk of 500. Approximately how many base pairs are in this DNA? How is the linking number altered (increases, decreases, doesn’t change, becomes undefined) when (a) a protein complex is bound to form a nucleosome, (b) one DNA strand is broken (c) DNA gyrase and ATP are added to the DNA solution, or (d) the double helix is denatured by heat?

Linking number

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Kornberg and his colleagues incubated soluble extracts of E. coli with a mixture of dATP, dTTP, dGTP, and dCTP, all labeled with 32P in the α-phosphate group. After a time, the incubation mixture was treated with trichloroacetic acid, which precipitates the DNA but not the nucleotide precursors. The precipitate was collected, and the extent of precursor incorporation into DNA was determined from the amount of radioactivity present in the precipitate.

(a)   If any one of the four nucleotide precursors were omitted from the incubation mixture, would radioactivity be found in the precipitate? Explain.

(b) Would 32P be incorporated into the DNA if only dTTP were labeled? Explain.

(c) Would radioactivity be found in the precipitate if 32P labeled the β or γ phosphate rather than the a phosphate of the deoxyribonucleotides? Explain.

DNA replication

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DNA polymerases are not able to prime replication, yet primase and other RNA polymerases can. Some geneticist have speculated that the inability of DNA polymerase to prime replication is due to its proofreading function. This hypothesis argues that proofreading is essential for faithful transmission of genetic information and that, because DNA polymerases have evolved the ability to proofread, the cannot prime DNA synthesis. Explain why proofreading and priming functions in the same enzyme might be incompatible?

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A number of scientist who study ways to treat cancer have become interested in telomerase. Why would they be interested in telomerase? How might cancer-drug therapies that target telomerase work?

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The enzyme telomerase is part protein and part RNA. What would be the most likely effect of a large deletion in the gene that encodes the RNA part of telomerase? How would the function of telomerase be affected?

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Some E. coli mutants contain defective DNA ligase. When these mutants are exposed to 3H-labeled thymine and the DNA produced is sedimented on an alkaline sucrose density gradient, two radioactive bands appear. One corresponds to a high molecular weight fraction, the other to a low molecular weight fraction. Explain.

Function of DNA ligase

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Suppose a future scientist explores a distant planet and discovers a novel form of double stranded nucleic acid. When this nucleic acid is exposed to DNA polymerases from E. coli, replication takes place continously on both strands. What conclusion can you make about the structure of this novel nucleic acid?

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In the presence of a eukaryotic condensin and a type II topoisomerase, the Lk of a relaxed closed-circular DNA molecule does not change. However, the DNA becomes highly knotted.

Formation of the knots requires breakage of the DNA, passage of a segment of DNA through the break, and religation by the topoisomerase. Given that every reaction of the topoisomerase would be expected to result in a change in linking number, how can Lk remain the same?

DNA topology

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The DNA below is replicated from left to right. Label the templates for leading strand and lagging strand synthesis.

(5')ACTTCGGATCGTTAAGGCCGCTTTCTGT(3') (3')TGAAGCCTAGCAATTCCGGCGAAAGACA(5')

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A suitable substrate for DNA polymerase is shown below. Label the primer and template, and indicate which end of each strand must be 3' or 5'.

To observe DNA synthesis on this substrate in vitro, what additional reaction components must be added?

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DNA synthesis on the lagging strand in E. coli is a complex process known to involve several proteins. Initiation of a new chain is catalyzed by the enzyme (a) _____________, and elongation is catalyzed by the enzyme (b)______________.

Synthesis is discontinuous, yielding short segments called (c) _______________, which are eventually joined by the enzyme (d)______________, which requires the cofactor (e)___________.

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What will be the effect on DNA replication of mutations that destroyed each of the following activities in DNA polymerase I?

a. 3’5’ exonuclease activity b. 5’3’ exonuclease activity c. 5’3’ polymerase activity

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