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bchm 218 part 1 > Module 2 Section 2 > Flashcards

Module 2 Section 2 Flashcards

1
Q

6 Principles of DNA replication

A
  1. Replication is semi-conservative
  2. Replication is initiated at specific sites
  3. Replication is typically bidirectional
  4. replication is semi-discontinuous
  5. RNA primases needed to start DNA replication
  6. nucleases, polymerases, ligases replace DNA primers with DNA and seal remaining nick
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2
Q

About sites of replication

A
  • linear chromosomes can have multiple origins

- circular chromosomes have one origin

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3
Q

Replication fork

A

point where parental duplex separates and daughter duplexes form

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4
Q

Replication bubble

A

open DNA being replicated

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5
Q

Bidirectionality of replication

A

-replication occurs toward and away from replication fork but always in 5’-3’ direction

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6
Q

About replication semi-discontinuity

A
  • leading strand synthesized continuously
  • lagging strand discontinuously in opposite direction of replication fork
  • Okazaki fragments produced
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7
Q

About primers

A
  • leading and lagging strand need 10-13 BP RNA primers to start replication
  • primers must be complementary to template and contain free 3’OH group
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8
Q

What is a nuclease

A

enzymes that hydrolyze the phosphodiester linkages of nucleic acids

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9
Q

Polymerases

A

-enzymes that catalyze template-dependent synthesis of DNA from its dNTP precursors

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10
Q

Ligases

A

enzymes that create phosphodiester bond between 3’ end of DNA segments and 5’ end of another

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11
Q

Domains of DNA Pol I.

A
  1. the DNA polymerase
  2. 3’-5’ proofreading exonuclease
  3. 5’-3’ exonuclease
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12
Q

Overall goal of exonuclease

A

enzyme that hydrolyzes only the phosphodiester bonds that are in terminal positions of the nucleic acid

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13
Q

Process of sealing nicks w DNA Pol I.

A
  • At the pick , Pol I. degrades RNA primer in 5’-3’ direction, releases dNTPs
  • simultaneously extends 3’ term with dNTP in same direction
  • nick moves in 5’-3’ direction until all RNA removed
  • Ligase seals fragments
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14
Q

DNA Pol Structure

A
  • resembles right hand
  • Fingers: where dNTP enter
  • Thumb: holds template DNA in place
  • Palm: DNA stand lays here, contains active site of enzyme
  • Pol I. must be in closed form for rxn to take place
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15
Q

Quality control

A
  • Pol. I relies on shape recognition
  • G-C and A-T have shapes that fit well in closed form of active site
  • incorrect pairs prevent full closure of Pol around DNA
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16
Q

Moieties

A

specific groups of atoms within a molecule responsible for characteristic chem. rxns if that mlc

17
Q

About Pol. active site

A
  • closed conformation allows important residues to contact sugar+phosphate moieties of incoming nucleotide
    1. Asp residues (coordinate 2 divalent Mg++ atoms)
    2. 1st Mg++ deprotonates 3’OH of growing strand, forms phosphodiester bond, releases pyrophosphate
    3. 2nd Mg++ binds -ve charge phosphate groups in incoming dNTP, facilitates rapid release of phyrophosphate LG after addition of nucleotide
18
Q

Steps of DNA Pol. Rxn

A
  1. Need primer strand, template strand, dNTPs
    - dNTP added to 3’OH of primer strand causing growth
  2. Pol. I moves forward to new 3’-term
  3. after dNMP added, new terminal BP occupies insertion site
    - must be translocated to postinsertion site
    - occurs by dissociation of enzyme from DNA followed by rebinding with terminal BP in postinsertion site
19
Q

Pyrophosphate

A

phosphorous oxyanions with 2 P atoms in P-O-P linkage (diphosphate)

20
Q

Insertion site

A

contains template strand nucleotide that will pair with incoming dNTP

21
Q

Postinsertion site

A
  • contains 3’-OH group of primer strand
  • 3’OH attacks alpha and beta phosphates of incoming dNTP, results in addition of dNMP to primer strand + release of pyrophosphate
22
Q

Translocation process of DNA Pol

A
  • can slide b/c of several + charge Lys residues in palm domain
  • interact with phosphodiester backbone
23
Q

Processivity #

A
  • the average # of nucleotides incorporated before enzyme dissociates from DNA
  • low processivity used for a few bases (Okazaki frag.)
  • high processivity used for long fragments
  • Pol I has low #
  • Pol III has high #
24
Q

3’-5’ exonuclease activity (DNA Pol)

A
  • when incorrect pair, Pol will fray DNA by 4 nucleotides
  • places incorrect dNMP in 3’-5’ exonuclease active site, cleaves incorrect dNMP
  • one Mg++ deprotonates H2O to form OH- nucleophile which mediates hydrolysis of incorrect dNMP (leaves free OH)
  • another Mg++ promotes departure of incorrect dNMP LG from growing strand

bchm 218 part 1 (11 decks)

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