Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BCHM 218 Part 1 > Module 2 Section 3 > Flashcards

Module 2 Section 3 Flashcards

1
Q

E.Coli Pol I features

A
  • 1 subunit
  • Okazaki fragment processing +DNA repair
  • 3’-5’ and 5’-3’ exonuclease
  • not used in chromosome replication
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

E.Coli Pol II features

A
  • 1 subunit
  • Translesion synthesis
  • 3’-5’ exonuclease
  • possibly used for DNA repair
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

E.Coli Pol III features

A
  • 3 subunits
  • 3’-5’ exonuclease
  • used for chromosome replication, sometimes called replicase
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

E.Coli Pol IV/V features

A
  • Pol. IV: 1 subunit, Pol. V: 2 subunits
  • no proofreading exonucleases
  • translesion DNA polymerases
  • used when DNA damage halts replication fork, get replication moving again
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

E.Coli replication stages

A
  1. Initiation of replication
  2. Elongation by the replisome
  3. Termination
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

DnaA 9-mer sites

A

4 copies of 9-nucleotide (9-mer)consensus sequence to which bacterial initiator protein DnaA binds

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

A=T rich 13-mer repeats

A
  • to one side of the DnaA 9-mer sites are 3 A=T rich direct repeats (13 BP each)
  • repeats known as DNA unwinding element, unwind readily upon binding of initiator
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Steps of oriC activation

A
  1. Generate open complex
  2. activate replication origin
  3. assembly of E.coli replication forks
  4. replication initiation and leading strand synthesis
  5. lagging strand synthesis
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Generation of Open complex

A
  • when bound to oriC, DnaA oligomerizes, + wraps DNA around oligomer complex (strains DNA)
  • in presence of ATP, DnaA destabilizes A=T rich 13mer repeats of oriC, forms ssDNA bubble with help of HU protein
  • complex formed is DnaA-ATP-oriC-HU (open complex)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

About DnaA

A
  • in AAA+ protein family
  • AAA+ family has ATPase activity, binds/hydrolyzes ATP structural domain that assist in protein + DNA conformational changes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Activation of Replication origin

A
  • ssDNA regions are exposed, 2 hexamers of DnaB helicase assemble ( 1 on each strand)
  • DnaC used to load DnaB by prying open ring of DnaB and slipping around ssDNA
  • ATP bound DnaC binds to DnaB, represses helicase ability
  • hydrolysis of ATP ejects DnaC from DnaB
  • protein-DNA assembly at the oriC called prepriming complex
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Assembly of E.coli replication forks

A
  • DnaB binds ATP, allows it to translocate+unwind DNA, dislodging DnaA protein
  • unwinding occurs outward in both directions, widens replication bubble
  • topoisomerase enzyme relieves supercoil stress
  • newly unwound DNA in bubble coated with SSB proteins
  • primase cannot interact with DnaB until bubble is 100-200 BP in size
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Replication initiation and leading strand synthesis

A

-RNA primer directs loading of beta clamp and assembly of leading strand Pol III holoenzyme

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Beta clamp

A
  • component of E.coli Pol. III holoenzyme
  • ring shaped homodimer encircles and slides along duplex DNA ahead of Pol III core (attached to Pol III)
  • enhances processivity
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Holoenzyme

A
  • active form of an enzyme

- enzyme that requires co-factor but is not bound to it is called apoenzyme

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Lagging strand synthesis in E.coli

A
  • Pol III holoenzyme extends its RNA primer until both Pol III complexes connect w DnaB helicase travelling in same direction on other side of bubble
  • priming is followed by clamp loading and synthesis of lagging strand by 2nd Pol III enzyme in the complex
  • this generates multiple Okazaki fragments discontinuously
  • replication continues until end of template of until another replication fork from an adjacent oriC is reached
17
Q

Initiation in Euk vs. E.coli

A
  • Euk have higher DNA content, slower replication forks
  • Euk need multiple origins of replications
  • Euk origins fire once per cell cycle
18
Q

S. cerevisiae replication (eukaryotic)

A
  • origins called ARS (autonomously repeating sequences), AT rich
  • indicator called origin recognition complex (ORC), subunits similar to DnaA, ATP required for ORC binding
  • after ORC binds to DNA, Cdc6 (AAA+) binds to ORC (similar to HU)
  • ORC-Cdc6 complex loads Mcm2-7 complex onto DNA
  • Mcm2-7 (DnaB-Like) is circular hexamer, binds to 1 mlc of Cdt1 (DnaC-like)
  • this is required before ORC-Cdc6 complex can load Mcm2-7 complex onto DNA
  • ABOVE EVENTS OCCUR ONLY IN G1 PHASE
  • resulting complex called prereplication complex (preRC)
  • after addition of proteins+checkpoints, RC begins to replicate DNA in S phase
19
Q

E.coli replisome

A
  • Pol. III holoenzyme, DnaB helicase, primase
  • helicase connects to Pol. III core through tau subunits of clamp loader
  • connection between DnaB helicase and Pol. III holoenzyme increases speed of DnaB
  • 3 tau subunits of tau complex clamp loader bind 3 Pol III cores (same tau subunits that bind DnaB)
  • leading strand Pol.III-B-Clamp complex moves continuously w DnaB
  • Lagging strand Pol. III-B-clamp complex repeatedly moves on and off DNA to extend multiple RNA primers made by primase
20
Q

B-clamps facilitate processivity

A
  • B-clamps ensure contact between Pol III core and DNA

- B-slidiing clamps assembled onto both DNA strands by one clamp loader

21
Q

Clamp loader mechanism

A

-in Pol. III holoenzyme is tau complex
-uses energy of ATP binding to open B-clamp
(can’t bind clamp w/o ATP)

22
Q

About Trombone Model

A
  • lagging strand polymerase extends 3’term of okazaki fragment in opposite direction to fork movement
  • pol. still part of replisome, moves with fork
  • results in DNA loop for each okazaki fragment
  • when lagging strand pol. finishes Okazaki fragment, it dissociates from DNA to transfer to a new RNA primer (detaches from B-clamp, associates w new B-clamp at next primer)
23
Q

Steps of Clamp recycling

A
  1. Pol. III core dissociates from B-clamp following synthesis of Okazaki fragment
    - B-clamp site attracts Pol. I, removes RNA primer w 5’-3’ exonuclease
  2. Pol. I dissociates from DNA, leaves ssDNA break
    - B-clamp attracts DNA ligase, forms phosphodiester bond to seal break
  3. unoccupied B-clamp opened + unleaded by excess delta subunits of clamp loader
24
Q

Termination of E.coli replication

A
  • region halfway around circular chromosome from oriC contains 2 clusters of 23 BP sequences (Ter sites, oriented in opposite directions)
  • monomeric Tus protein binds to Ter site, blocks advance of replication fork by stopping DnaB helicase
  • Tus-Ter complex is directional, replication forks only blocked when approaching complex from nonpermissive direction
25
Q

Competition between transcription + replication in E.coli chromosome

A
  • replicating bacteria is still transcribing RNA from promoters
  • collisions between RNA pol and replication forks happens
  • codirectional collisions do not impede fork
  • head on collisions cause fork to pause/stall
  • most collisions codirectional
26
Q

Steps of topoisomerase mechanism

A
  • type II topoisomerase in prokary is heterodimer, 2 ATPase domains, 2 cleavage core domains
  • pulls one piece of catenated (tangled) dsDNA, cleaves with ATP (at cleavage site using Tyr residue)
  • Tyr gives up proton to form nucleophile, attacks phosphate in phosphodiester bond
  • temporarily covalently links DNA to protein
  • once daughter chromosomes separated, hydrolysis rxn reforms phosphodiester bond
27
Q

Problem with eukaryotic termination

A
  • at end of chromosome, once leading strand completely extended, RNA primer at the extreme end needs to be replaced with DNA
  • PROBLEM: no 3’ term. for the Pol. to extend from, so genetic info in the gap would be lost in next round of replication
  • progressive shortening after multiple rounds of replication
28
Q

Solution to eukaryotic termination problem

A
  • ends of euk chromosomes called telomeres (repeats of a unique sequence)
  • telomerase reverse transcriptase (TERT) carries non-coding telomerase RNA (TR)
  • TR contains 1.5 telomerase repeats, used as template to extend 3’ term. of telomere beyond what was replicated
  • TERT-TR holoenzyme is called telomerase
  • RXN OCCURS IN S PHASE
29
Q

Telomerase process steps

A
  1. @ 3’-term, 3 nucleotides of telomere anneal to 3 RNA nucleotides on telomerase
  2. Telomerase extends 3’ end of ssDNA by length of one telomere repeat
    - telomere carries own template, synthesizes ssDNA
  3. after adding telomere repeat, telomerase separates from DNA-RNA hybrid
    - repositions on telomere for next extension, acts processively
  4. telomerase extended 3’ ssDNA term. converted to dsDNA by same priming/polymerization machinery used in chromosome replication

BCHM 218 Part 1 (11 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions