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MC Biochem 1 > Chapter 28 > Flashcards

Chapter 28 Flashcards

1
Q

DNA structure

A

B form- Double Helix:
Diameter of helix-20A
10.4 BP per turn
3.4 A between BP

1) unbranched (linear) polymer of monodoxynucleotides

2) 3’ to 5’ phosphodiester bonds which imparts uniform negative charge to DNA/RNA
- negative charge repels nucleophilic species thus the phosphodiester bond is resistant to hydrolytic attack

3) complementary base pairing connected by hydrogen bonding A-T (2) and G-C (3)
- Hydrophobic interactions due to base stacking

4)antiparallel strands; 5’ pohosphate and 3’ free OH

5)has major and minor grooves
-allows specific interaction with proteins through hydrogen bond donor atoms and acceptor atoms
Major groove: Wider and deeper
-more accessible for interactions with proteins
Minor Groove-Narrower and shallower

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2
Q

3 forms of DNA

A

A form

  • shorter and fatter
  • dehydrated
  • ribose is C-3’ ends
  • also found in double stranded RNA
  • R handed

B form (normal form-watson and crick)

  • longer and narrower
  • hydrated
  • ribose is C-2’; 2’ carbon lies out of the plane of other atoms
  • form found most often in vivo
  • right Handed

Z form: (zig zag)

  • alternating sequences of purines and pyrmidines
  • 180 degree flip BP
  • Left handed
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3
Q

Watson and Crick

A 
Determined to structure of DNA by:
1) X-ray diffraction photograph of DNA crystals
-Maurice Wilkins and Rosalind Franklins
-2 chains formed a helical structure
2) Chargraff's Rule
-Edwin chargraff determined the composition of DNA
-[A]=[T]
-[G]=[C]
3) Bond Angles in Reference Books
Complementary base pairing
4) Built Models
-nucleotide content determines DNA melting point or number of hydrogen bonds
G to C has 3 h bonds
A to T has 2 h bonds

Nobel Prize in 1962 of Physiology or Medicine
Maurice Wilkins, Francis Crick, James Watson
DNA structure

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4
Q

Replication Fork

A

1) Formed by unwinding helix
2) moves bidirectionally from origin as synthesis occurs
- Fork is initiated by Pre-priming complex: DnaA protein, SSB and helicase
- synthesis: primase, DNA polymerase, exonuclease

3) Unwinding creates strain on region just beyond replication fork
- control of topology is Topoisomerase

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5
Q

Helicase

A

Unwinds DNA

  • binds near origin or replication fork to one strand of DNA and seperates the strands by breaking H bonds thus unwinding the DNA
  • requires ATP (energy)
  • DONT BIND BOTH STRANDS AND PULL APART

PcrA-bacterial helicase;

  • composed of 4 subunits
    1) A1
  • contain P loop for binding and hydrolysis of ATP
  • bind ssDNA
    2) A2
  • unique domain
    3) B1
  • homologous to A1 but lacks P Loop
  • binds ssDNA
    4) B2
  • unique domain

Alignment of numerous helicases demonstrate similarities to A1 and B1

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6
Q

SSB

A

Single Stranded DNA binding protein

1) Helix stabilizing protein
2) structural proteins
3) Coopoeratively binds ssDNA (High affinity for)
- prevents reannealing of complementary bases, which allows DNA polymerase to read bases
- protects single stranded regions by resisting neclease digestion

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7
Q

Cell Cycle

A

Eukaryotic

1) Synthesis (DNA replication)
2) G2=gap 2 (getting everything ready for cell division)
3) Mitosis-cell division
4) G1= Gap 1 (warming up getting ready for S)

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8
Q

Origin of replication

A

Start site of replication, with this process DNA strands separate or “melt”

origin or ori:
1 in Prokaryotes
-genome is a single, circular, double stranded, DNA molecule (chromosome)
E. coli- ori C; 
-1 ori for 4.6 million BP
 245 bp region
-three 13mer tandem repeats (AT rich region)
-five binding sites for DnaA

Numerous in eukaryotes

  • genome consists of many, linear, double stranded DNA molecules
  • high [AT} base pairs leads to low melting point
  • humans have 30,000 oris to replicate to 6 million BP
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9
Q

Helicase Mechanism

A

MECHANISM

1) A1 and B1 of helices bind ssDNA
2) ATP binds to the P loop on the A1 subunit causing a conformational change
- A1 releases DNA and slides closer to B1 (cleft closes)
2) Hydrolysis of ATP to ADP and Pi causes a conformation change
- DNA is pulled across B1 toward A1 and helicase translocates along DNA causing the cleft to OPEN
3) helicase moves along one strand displacing the other strand

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10
Q

DnaA Protein

A

Binds to and melts origin of replication

  • 20 to 50 copies/origin
  • origin is AT rich: low Tm

identifies the origin of replication in E. coli

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11
Q

Super Coiling

A

260 BP long DNA molecule

  • Right handed helix
  • 25 turns of helix (1 turn per 10.4 BP)

Lk=Tw+ Wr
Lk=linkage number
Tw=twist, measure of winding of strands around each other
Wr=Writhe, measure of supercoiling
Change in linking number usually expressed
-70% writhe
-30% twist

Supercoild:
Right handed-negative
-Negative supercoiling prepares DNA for unwinding
Left handed-positive

Topological isomers or Topoisomers

The amount of supercoiling affects rate of migration through an agrouse gel

  • supercoiled- faster
  • open circular-slower
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12
Q

Topoisomerase

A

Relieves torsional strain in DNA molecule (alters topology of DNA molecules)

  • Breaks phoshodiester bonds by nuclease activity
  • Reform phophodiester bonds by ligase activity-strand resealing

Topoisomerase I
Topoisomerase II

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13
Q

Topoisomerase I

A

Function:

  • relaxes supercoiled DNA by breaking phosphodiester bond on ONE strand of double helix
  • DNA and topoisomerase form covalent bod and tyrosine acts as nucleophile

Contains 1 Cavity

Mech:

1) DNA binds inside cavity
2) Nucleophilic attack on phosphate by hydroxyl of try, which releases 5’ hydroxyl creating a covalent intermediate with phosphate
3) Rotation around intact DNA strand due to energy from supercoiling
4) Free hydroxyl of DNA attacks the phosphotyrosine residue and tyrosine is released

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14
Q

Camptothecin

A

Antileukemic and Antitumor Drug

  • alkaloid derived from the bark of tree Camptotheca acuminate
  • inhibits topoisiomerase I and can be used to treat cancer
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15
Q

Topoisomerase II

A

3 functions; relaxes, supercoils, decatenate
Relaxes or supercoils DNA by breaking phosphodiester bonds on BOTH strands of double helix
-ATP required for supercoiling

Functions as a DIMER

  • contains 2 cavities for binding DNA
  • each subunit binds ATP

BINDS two segments:

  • G segment-gate segment
  • T segment-transport segment
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16
Q

decatenation

A

Function of TOPO 1 not TOPO 2

unlinking of chains and relinking to form 2 separate links

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17
Q

Etoposide

A

Chemotherapy
-Used to treat multiple forms of cancer
Inhibits topoisomerase II

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18
Q

Novobiocin

A

Antiobitc

  • use to treat gram positive bacteria
  • inhibits ATPase activity of DNA Gyrase
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19
Q

DNA Gyrase

A

specific type of bacterial Topoisomerase II that supercoils, relaxes, and decatenate DNA

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20
Q

Ciprofloxacin

A

Antibiotic

  • used to treat gram-positive and gram-negative bacteria
  • inhibits DNA gyrase by breakage and resealing activity (ligase activity)
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21
Q

Primase

A

Type of RNA polymerase that catalyzes de novo synthesis of RNA primer complementary to DNA

RNA Primer:

  • 5 to 10 nucleotides long
  • creates RNA:DNA duplex
  • provides “free” 3’ OH which is required by DNA polymerase
  • removed later in reaction
22
Q

DNA polymerase

A

Class of enzymes that synthesizes DNA by addition of nucleotides in a template directed fashion- 5’ to 3’

  • template is read 3’ to 5’
  • Prokaryotic and Eukaryotic DNA polymerases
  • five structural classes known

DNA polymerase interacts with nucleotide in Minor grooves:

  • Arg and Gln residues of DNA polymerase H bond with Basepairs to determine if base pair is the proper width
  • if adds wrong purine (AG) base=wider
  • if adds wrong pyrimidine (TC) base=narrower

Requires:

1) template DNA
2) 3’ hydroxyl-is the initial 3’ hydroxyl provided by RNA primer which is synthesized by primase
3) dNTPs
4) Mg2+ (or other divalent cations)

23
Q

DNA replication: Mg2+

A

Requirement for DNA polymerase
-not present in active site, because Mg2+ brings nucleotide to the active site

2 Mg2+’s in reaction:

  • 1 interacts with the phosphate of dNTP and 3’ OH of the primer activating it
  • other interacts with another different phosphate of dNTP

They both are bridged by the R group (carboxylate) of 2 Asp which stabilizes the pentacoordinate intermediate

24
Q

Prokaryotic DNA polymerase

A

Named using roman numerals FOUND IN E. COLI

  • DNA polymerase I= repair/gap filling
  • DNA polymerase II- repair
  • DNA polymerase III- replication
25
Q

Eukaryotic DNA Polymerase

A

Named using greek alphatabet (FOUND IN HUMANS)

1) alpha DNA poly
- contains primase
- initiation
- replicate DNA
- NO proofreading capability

2) Beta DNA poly
- repair DNA
- no proofreading capability

3) Gamma DNA poly
- mitochondrial DNA replication
- HAS proofreading capability

4) Delta DNA Poly
- leading and lagging strand
- replicate DNA
- HAS proofreading capability

5) Epsilong DNA polymerase
- -repairs DNA
- HAS proofreading capability

26
Q

Characteristics of DNA polymerase

A

Processivity

  • # of nucleotides incorporated for each association of enzyme with the template/primer
  • replication=high (ex: E.coli DNA poly II)
  • repair=low
  • increased Bly sliding clamp

Fidelity

  • measure of accuracy in how many base pairs are wrong?
  • high fidelity=few errors
  • low fidelity=many errors
  • Increased by 3’ to 5’ exonuclease (proofreading)
27
Q

Sliding Clamp Protein

A

Locks DNA onto DNA polymerase (E. coli DNA poly III or B2)

increaseses processivity

DNA is loaded into sliding clams by clamp loads and requires ATP

28
Q

Nucleases

A

Digest DNA-remove errors/BP
Two types:
1) Exonuclease
-digests DNA from one end
-5’ to 3’ digests DNA in front of synthesis/replication
-3’ to 5’ (proofreading) removes one newly synthesized nucleotide

2) endonuclease
- cleave internal phosphodiester bonds producing single stranded nicks
- restriction endonucleases- Ex: EcoR V

29
Q

3’ to 5 exonuclease

A

Proofreading

  • removes incorrectly incorporate nucleotides (opposite direction of DNA polymerase)
  • activity of many DNA polymerases
  • increase fidelity of replication
30
Q

5’ to 3’ Exonuclease

A

Digest DNA and RNA ahead of DNA synthesis
-hydrolytically removes RNA primer ahead of synthesis

Activity of DNA polymerase I
-leaves one nick when it removes RNA primer and replaces with DNA

31
Q

Structure of DNA polymerase specifically E. coli DNA poly III

A

Holoenzyme
Central structure-Helicase bound to Clamploader protein

2 copies of core enzyme:

  • alpha- DNA polymerase
  • beta-2 copies slidng clamp
  • epsilon-3’ to 5’ exonuclease
  • sigma
32
Q

3 Forms of DNA

A

B Form-(normal form-watson and crick)
Right Handed
-hydrated form

A form:

  • dehydrated form
  • nucleotides tilted 20 degrees relative to helical axis
  • Right handed

Z Form (zig zag form)

  • stretches of alternating purine and pyrimidines
  • base pairs flip 180 degrees
  • L handed helix
33
Q

What holds DNA together?

A

DNA is held together by:

1) Hydrogen bonding between base pairs
2) Hydrophobic interactions (Van Der Waals) due to base stacking

34
Q

Semiconservative Synthesis

A

-each strand serves as a template for synthesis of a new complementary strand

35
Q

DNA Ligase

A

Seals Nicks

  • synthesizes phosphodiester bonds between the 3’ free OH and the 5’ phosphate (at opposite ends)
  • requires ATP for archaea and eukaryotes
  • requires NADP+ for bacteria
36
Q

DNA replication in E.coli

A

1) The binding of DnaA proteins to OriC on DNA
2) Single DNA strands are exposed to the prepriming complex (Helicase, SSB, and DnaA)
3) The DNA polymerase III holoenzyme assembles on the prepriming complex and begins synthesizing

37
Q

Trombone Model

A

1) Lagging strand template is looped out so DNA poly III synthesizes in same direction as leading strand
2) DNA poly III releases lagging strand template after adding 1000 nucleotides by releasing sliding clamp
3) New loop is formed, sliding clamp is added and primase synthesizes a RNA primer to initiation the formation of another okazaki fragment segment’
4) The gaps between fragments are filled by DNA poly I, and also removes RNA primer (5’ to 3’ direction)
5) Finally DNA ligase connects fragments

Slide of trombone lengths as you add nucleotides

38
Q

Telomerase

  • def
  • mech
A

Specialized eukaryotic (humans) reverse transcriptase

  • ribozyme
  • synthesizes the linear end of chromosomes (telomeres)
39
Q

Chromosome Parts:

A

1) centromere-site that connects sister chromatids
2) Kinetochore-site where the spindle fibers attach to chromosome

3) Telomere-nucleotide repeat at end of linear chromosome
- synthesized by telomerase
- humans have AGGGTT repeat
- protects end from nuclease digestion
- can be used to tell someones genetic age-shorter over the years

40
Q

Telomere structure in vivo

A

ss region of DNA “invades” and H-bonds to telomere repeat sequence
-stabilized by telomere binding protein

41
Q

Human immunodeficiency virus (HIV)

A
  • retro virus

- uses reverse transcriptase to convert RNA (RNA template) into viral DNA which integrates into human genome

42
Q

Reverse Transcriptase Inhibitors

A

1) Nucleoside analogues-chain terminators
- 2’3’dideoxyinosine(ddI or Didanosine)-lack 2’ and 3’ Hydroxyl of sugar thus not nucleophilic attack
- AZT

2) non nucleotide analogues
3) Protease inhibitors

43
Q

DNA repair

A

repair is due to damage that occurs from various sources

  • Strand-directed mismatch repair
  • repair of Damage of UV light
  • base excision repair
  • repair of double stranded breaks
44
Q

types of DNA damage

A

1) Mismatch base pairs-replication error
- Transition-Nucleotide substitutes for same type
- Transversion-nucleotide substitutes for different type
2) insertion/deletions
- insertions of 1 or 2 nucleotides alter reading frame of mRNA and alter amino acids that are going to be transcribed
- deletion of regulatory info-bad

45
Q

DNA damaging agents found in vivo

A
  • Nitrous acid
  • radiation
  • ultraviolet light
  • high energy radiation
46
Q

Repair of Damage of UV light

  • damage to?
  • mech
  • disease?
A

Damage causes Thymine Dimers which prevents replication and transcription

1) use UV specific endonuclease-UVR ABC endonuclease which cleaves 5’ to 3’ dimers
2) DNA polymerase replaces the nucleotides
- e.coli DNA poly 1 if prokaryotic cell
3) DNA ligase seals the nick

Disease state:
Xeroderma pigmentosum
-cells cannot repair DNA damagae
-mutations accumulate=cancer
-absence of UV-specific endonuclease
47
Q

Base excision repair

  • damage to?
  • mech
  • disease?
A

Damage=altered bases; deamination of cytosine to uracil

Mech:

1) Uracil DNA glycosylase removes abnormal base and creates AP-site (apurinic or apyrimidic)
2) AP endonuclease cleaves
3) deoxyribose-phosphate lyase
4) DNA polymerase
5) Ligase

48
Q

Repair of double stranded breaks

  • Damage to?
  • mech?
  • disease
A

Many cuases-potentially lethal

Two types

1) Nonhomologous end joining repair
2) Homologous recombination repair

49
Q

DNA polymerase Reaction mechanism

A

SAME AS RNA POLYMERASE FOR TRANSCRIPTION

1) Nucleophilic attack by the 3’ OH (provided by the RNA primer) on the alpha phosphate of dNTP
2) Pyrophosphate (PPi) is hydrolyzed to orthophosphate (Pi+Pi)

50
Q

Topoisomerase II Mechanism

A

1) binds DNA strand-G strand tightly
2) Nucleophilic attack by the OH of tyrosine forms a covalent bond to each DNA strand (2)
3) binds second DNA strand-T segment loosely
4) ATP binds and triggers a conformational change bring the two ATP domains together which traps the T segment and cleaves both strands go G segment
5) Religation of G segment causes T segment to be released out the bottom gate of the enzyme
6) Hydrolysis of ATP and release ADP and orthophosphate (2Pi) causeless a conformational change and the enzyme resets

OVERALL process Leads to a decrease in linkage Number BY2

MC Biochem 1 (30 decks)

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