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Genetics > DNA Replication/Repair/Recombination (lecture 6) > Flashcards

DNA Replication/Repair/Recombination (lecture 6) Flashcards

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1
Q

eukaryotic ARS

A

synonyme for origin of replication?

Replication is linked to the cell cycle (S-Phase)

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2
Q

when are nucleosomes synthesized?

A

nucleosome is histone-octamer (H2A, H2B, H3, H4, synthesized

during S-phase unlike most other proteins)

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3
Q

nucleosome structure

A

2x H2A, H2B, H3, H4 (octamer)

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4
Q

problem with nucleosomes in replication? solution?

A

Steric obstacle for the replication fork

Chromatin remodeling proteins destabilize DNA-histone interactions

  • > H2A-H2B dimers dissociate
  • > H3-H4 parental tetramers remain DNA associated, randomly segregate to one of the new strands

new nucleosomes are mostly hybrids of new and old histones

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5
Q

histone chaperones?

A

NAP/CAF (chromatin assembly factors)

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6
Q

replication and epigenetics?

A

as a result of the random re-distribution old of histones during replication, approx. one half one half of the daughter nucleoseomes have modified histones

to maintain the epigenetic code, the reader-writer-complex catalyzes the same modification it writes

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7
Q

telomerases? function?

A

in most cells activity can not be detected

extend the 3’ end by RNA-templated DNA synthesis

synthesis of repeat sequences ensures that telomerase can always bind and always elongate

special feature: RNA template!

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8
Q

how can repair mechanisms distinguish between ds breaks and chromosome ends?

A

T-loop of protruding 3’ end
After lagging strand synthesis, 5’ end is shortened to produce 3’ overhang (to ensure free ss for
further elongation by telomerase).
3’ overhang loops back and inserts into the double strand

-> protection against nucleases

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9
Q

Dolly

A

Edinburgh in Schottland wurden 277 Eizellen (Scottish Blackface) mit Zellkernen aus den Euterzellen des Spendertiers, Finn Dorset, geimpft.

Tod nach halber lebenserwartunge. Hypothese mgl. wegen Alterserscheinungen: Herkunft aus bereits gealterten adulten Zellen? ohne Telomerase Aktivität?

kein echter Klon da mitochondriale DNA aus Eizelle des Spender-Tiers

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10
Q

experimental change of telomere lenght

A

Experimental change
of telomere length
is readjusted in living cells

In some cell culture systems increasing telomere length can provide increased poten8al to divide

Telomerase ko mice develop cancer and age prematurely: cells divide in the absence of
telomerase ac8vity which results in disease due to unstable chromosomes

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11
Q

name reasons for random DNA changes

estimated rate of mutations which stay in genome?

A 
environmental influences (heat, radia8on, chemicals) and
metabolic accidents

1/1000 mutations stay

Muta8ons that affect DNA repair lead to increased muta8on rates and cause disease
->importance of DNA repair systems

many cancer diseases are due to broken DNA repair systems?

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12
Q

spontaneous changes in DNA?

A

Depurina8on: loss of G, A base due to hydrolysis; 5000/cell/day

Deamina8on of C (->U); 100/cell/day

Deamination of A, G
-> unnatural DNA bases

Oxida8on

Uncontrolled methyla8on

mutation because of methylation in transcription regulation:
spontaneous deamination from methylated C to T

-> result: deletions, base-pair substitutions

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13
Q

what happens to unnatural DNA bases?

how come?

A

can be detected

deamination:

A -> hypoxanthine
G -> Xanthine
Cytosine -> Uracil

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14
Q

T-G mismatches

A

detected by repair system

repair is biased to exchanging T for C-> restoring of the correct sequence
(why C needs to be biased? in the ds the sequence information is still stored?!)

but still: repair is ineffective: about 3% of C are methylated, mutations in these nucleotides account for about 30%
of single-base mutations in inherited human disease.

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15
Q

most frequent DNA damages7changes?

A

depurination

deamination

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16
Q

advantage of DNA double helix?

A

Double-helical structure of DNA allows repair as long as only one strand is affected
The complementary strand provides the template for the repair.
All genomes are ds, except some small viral genomes (ssDNA or ssRNA)

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17
Q

base excision repair

A

is responsible when a base is missing e.g. because a uracil DNA glycosylase has cut off a wrong U-base! (not the backbone)

  • AP endonuclease cleaves the back bone
  • DNA pol beta adds a new nucleotide
  • DNA ligase seals the nick (in the sugar backbone?!)
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18
Q

problem with base excision/ base changes

A

problem is no that sequence couldn’t be repaired (ds!)

problem occurs, if the mistake isn’t recognized and not repaired (ineffective repair system)

19
Q

UV-radiation can cause?

A

Thymin dimers or C-T dimers = pyrimidine dimers (covalent binding between two adjacent bases)

problem: blocks DNA replication and transcription

20
Q

solution for bulky lesions

A

= pyrimidine dimers

  • excision nuclease cuts the whole sequence around (12bp)
  • DNA helicase
  • DNA Pol and DNA ligase
21
Q

a reason for error prone reapair?

A

Heavily damaged DNA (stalled DNA replica8on) may ac8vate ‘rough and ready’
DNA repair systems (eu) (E.coli: SOS repair system), where special DNA polymerases
insert nucleo8des with less stringency (no 3’->5’ exonuclease ac8vity, inser8on of random
nucleo8des) . This results in increased muta8on rates.

-> aim is to revert the damaged DNA into a replicable state at all cost.
otherwise you loose the whole information

22
Q

distinguish between parental and new strand?

A

eu: nicks in new
pro: methylation in parental strand

23
Q

how transcription and DNA repair are coupled?

A

Transcrip8on-coupled repair (TCR):

while RNApol is stalled, specific repair proteins are
attracted to the transcrip8on bubble, repair the template strand (without displacing
the RNApol) and afterwards let transcrip8on resume(fortsetzen).

24
Q

repair and progression of the cell cycle are coupled?

A

DNA damage can:
-block entry from G1 into S phase (replication)

  • slow S phase
  • block transition from S into M phase

-> check points for cell cycle progression. This
gives the cell 8me to repair the DNA and prevent segrega8on of damages to
daughter cells.

25
Q

ways to repair ds breaks?

A

1) non-homologous end joining:

  • sticky ends are degradated to blunt ends
  • end joining
  • > DELETION of 1 or more nucleotides
    2) homologous recombination
  • formation of blunt ends (end processing)
  • information of sister chromatide is used to repair accurately
26
Q

when NHEJ is used?

A

common repair for soma8c cells
Most of the genome is non-coding
-> Small errors do not maler in most cases

27
Q

CRISPR/Cas9. abbreviation?

A

Clustered Regularly Interspaced Short Palindromic Repeats

originally: Bacterial acquired immune defence

application:
introduce ds brake -> nonhomologous end joining
-> dele8on (muta8on)

28
Q

functions of HR?

A

• Repair of ds breaks
• Exchange of genetic information between chromosomes
-> creating new genetic combina8ons (genotypes)

Evolu8onary conserved mechanism: also present in bacteria

29
Q

which principles are the basis for HR?

A

-strand invasion
- watson-crick base pairing = hybridization between homologous=similar (not identical!) sequences
does not require energy or enzymatic catalysis

30
Q

application of hybridization in the lab?

A
  • Microarrays

- hybridiza8on of probes to DNA immobilized on membranes or in tissue

31
Q

stabilization of ssDNA for hybridization during HR?

A

In vivo: ssDNA have to be stabilized for base pairing of homologous strands

RecA/Rad51: binds in long coopera8ve clusters to ssDNA
-> nucleoprotein filament
can at the same 8me interact with a dsDNA molecule

(ATP dependent)

32
Q

reaction during hybridization in homologous recombination?

A

Mul8step DNA synapsis reac8on:
RecA intertwines ssDNA with dsDNA molecules (sequence independent)
ssDNA ‘searches’ for homology
Strand invasion: ssDNA displaces the respec8ve strand of the double helix
-> hetero-douplex forma8on

33
Q

branch migration durin HR?

A
  • spontaneous: not directional

- protein-directed: specific direction mediated by helicase (ATP!)

34
Q

steps of HR after ds break?

A
  • exonuclease degrades 5’ ends
  • strand invasion mediated by RecA/Rad51 recombinases
  • branch migration (mediated by heliase) and DNA Pol
  • DNA ligation of one! ss, the other one is synthesized on this template

loss of heterozygosity

35
Q

similar mechanism as HR in ..? difference?

A

meiotic homologous recombination

  • ds break is introduced by spo1
  • Reciprocal strand invasion:
    cross-strand exchange
    or Holliday junc8on
  • both strands are synthesized at the template of the sister chromatide
  • in 10 % of the cases: cross over
    90% no cross over

(resolution of holliday junction determines)

36
Q

which proteins promote the branch migration in crossing over?

A

(meiotic HR)

RuvA and RuvB

37
Q

heteroduplex can lead to?

A

gene conversion = Gene conversion is the process by which one DNA sequence replaces a homologous sequence such that the sequences become identical after the conversion event.

because mismatch repair excises one strand (of the two nor properly paired strands)

-> one allele is lost

38
Q

role of mismatch repair in HR?

A

Components of the mismatch repair system prevent recombina8on between poorly matched sequences

39
Q

HR as a tool for genetic manipulation?

A

Bring the template DNA (containing homologous sequences and the target DNA) into the nucleus (injec8on, electropora8on)
Select for the cell where homologous recombina8on took place (selectable
marker: an8bio8cs resistance, fluorescence). Rare event, es8mate: 0.5x10^6/cell

Make an organism of the cell with the manipulated genome, -> requires to8potent cells
-> embryonic stem cells (ES cells)

40
Q

how can you assure that the integration of traget DNA was specific instead of random?

A

clone viral thymidine kinase beside the homologous sequence:
can convert gancyclovir
into cell toxic drug (chain termina8on during replica8on)

41
Q

tool to introduce ds break before HR?

A

CRISPR/Cas9

42
Q

other tool than HR for recombination?

A
  • Conserva8ve site specific recombina8on

• requires specific sequences (target sites for the recombinases)

• requires specific enzymes (recombinases); resemble topoisomerases I since they form
transient covalent bonds with the DNA. Require no energy. Reversal of the integra8on (excision)
restores original sequence (-> conserva8ve).

  • Phage lambda genome integration into e.coli
  • cre/lox system
43
Q

explain cre/lox system and application

A

loxP sites: inverted repeats which are placed up- and downstream of the target gene/exon

  • cre recombinase: recombinase which cuts out everything in between the loxP sites if it is expressed
  • combination of cre gene with a cell-type-specific promotor allows conditional knockouts in mice

Genetics (10 decks)

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