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BMS1062- > DNA Recombination: Double Stranded Breaks > Flashcards

DNA Recombination: Double Stranded Breaks Flashcards

1
Q

Double stranded breaks

cause and problem

A

Both strands are broken
• Problem: no template for repair
• Causes
• Environmental damage:
• Double stranded breaks can occur from ionising radiation (e.g. x-rays) and reactive chemicals (e.g. anti tumour-agents)
• DNA replication:
• Most of the time they arise from DNA replication forks that become stalled or broken (accidents occur during nearly every round of DNA replication).

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2
Q

what happens if Double stranded breaks is not repaired ?

A

If lesions not repaired:
• breakdown of chromosome into smaller fragments
• catastrophic loss of genes when cell divides

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3
Q

Double stranded break repair

Two mechanisms

A
  • Non-Homologous End Joining (NHEJ)
  • Re-joining of broken ends by DNA ligase
  • Generally loss of nucleotides at site of joining
  • Common in mammalian somatic cells
  • Homologous Recombination (HR)
  • More accurate
  • Uses sister chromatid as template
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4
Q

Double stranded break repair should occur between:

Select one:

a. Recombinant Chromosomes
b. Sister chromatids
c. Non-sister chromatids
d. Homologous Chromosomes

A

Double stranded break repair via homologous recombination (B) occurs between sister chromatids, using the undamaged chromatid as the template to repair the damaged chromatid

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5
Q

Non-homologous end joining

A
  • Ku protein: a heterodimer that grasps the broken chromosome ends
  • Protein complex forms, holding the two ends together
  • These proteins, including nucleases process the ends of the DNA
  • DNA polymerase may be required to fill any gaps
  • DNA ligase completes the process resulting in the end being covalently joined
  • Results in a loss of nucleotides at the site of joining
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6
Q

Homologous recombination

Introduction

A
  • Homologous Recombination involves the exchange of DNA strands between two homologous duplexes of DNA
  • Duplexes: double helix DNA
  • Homologous: identical or in this case, highly similar sequences
  • Sister chromatids (repair)
  • Homologous chromosomes (meiosis)
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7
Q

DNA base pairing: critical for recombination

A

Homologous recombination only between DNA duplexes that have extensive regions of sequence similarity
• Two DNA duplexes engage in extensive base-pairing between a single strand from one DNA duplex and the
complementary single strand from the other duplex
• A perfect match is not necessary but it must be close for homologous recombination to happen

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8
Q

Homologous recombination: functions

A
  • Accurate repair of double stranded breaks: most widespread use of homologous recombination
  • Genetic exchange between two homologous DNA sequences (DNA sequences similar or identical in nucleotide sequence)
  • Mechanical role in assuring accurate chromosome segregation during meiosis in eukaryotes
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9
Q

Double stranded break via HR

A

• Exonuclease chews/resects 5’ ends (cleaves nucleotides)
• Strand exchange/invasion by homologous base
pairing
• Extension of invading strand by DNA polymerase
• Invading strand released, original helices reformed
• Gap filling by DNA polymerase
• Ligation

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10
Q

Double stranded break repair during replication

A

• Nick or gap in parental DNA helix ahead of the replication fork
• When fork reaches lesion, it falls apart: causing a DS break in one duplex,
replication halts
• Homologous recombination restores DNA sequence using undamaged duplex as
template.
• Replication can resume

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11
Q

When does double-stranded break repair via homologous recombination occur?

Select one:

a. Before DNA replication
b. After DNA replication
c. Either before or after replication

A

Double stranded break repair via homologous recombination (B in the figure below) is very accurate. This is because it occurs shortly after DNA replication when the undamaged daughter DNA duplex or sister chromatid can serve as the template for repair of the damaged one.

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12
Q

How does a strand invade?

A

Invading strand needs a way of disrupting a stable DNA helix
• The 3’ end of the invading strand is acted upon by RecA (E. coli)
or Rad 51 (eukaryotes)
• ATP bound RecA binds tightly to ssDNA forming a DNAprotein filament
• RecA filament can hold a single strand and a double helix
• Catalyzes DNA synapsis between a DNA double helix and
a homologous region of single-stranded DNA
• Destabilises the duplex DNA

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13
Q

Process of strand invasion

A

• RecA protein intertwines the DNA single strand and DNA duplex in sequence independent manner
• DNA single strand searches duplex for homologous sequences
• Once homologous sequence located (extended stretch of at least 15 nucleotides), strand invasion occurs
• Single strand displaces one strand of the duplex and forms base pairs with the other strand resulting in a heteroduplex
(pairing of two DNA strands from two different DNA molecules
• Other proteins help in this process

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14
Q

Heteroduplexes

A

Some mismatch may occur
• Strand invasion requires the pairing of a region of single-stranded DNA with a complementary strand in a different DNA double helix
• DNA hybridisation occurs between strands that are highly similar, but not necessarily identical
• Creates a region of DNA helix formed from strands that originate from two different DNA molecules – heteroduplex
• Some mismatch occurs

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15
Q

What can go wrong in Homologous Repair?

A

Wrong template used (other chromosome homolog, not sister chromatid) lead to loss of heterozygosity
• Mutations can become homozygous
• Can lead to cancer eg. tumour suppressor genes
Too little homologous recombination = increased mutation rate - can lead to cancer
• Mutations in Brca1 and Brca2 cause breast cancer due to inefficient repair by homologous recombination
• Brca2 helps to bring Rad51 protein to sites of damage and releases it in its active form onto ssDNA

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16
Q

Mitosis

A

• Process of cell duplication or reproduction
where one cell gives rise to two identical
daughter cells
• Begins with one diploid cell containing two
copies of each chromosome
• Produces two identical diploid cells each
with two copies of each chromosome

17
Q

Meiosis

A

• Special type of cell division necessary for sexual reproduction
• Produces gamete cells
• Begins with one diploid cell containing two
copies of each chromosome
• Produces four haploid cells containing one
copy of each chromosome

18
Q

when dsDNA repair occurs

A

dsDNA repair occurs shortly after DNA replication in the S and G2 cell cycle phases - when a daughter duplex can act as
the repair template, ie. Before Mitosis

19
Q

Meiosis differs from mitosis in that:

A
  • Two divisions,
  • Homologous chromosomes separate
  • Sister chromatids separate
  • Chromosomes in meiosis undergo recombination
  • Shuffles the genes
  • Different genetic combination in each gamete
  • Chromosomes in mitosis are identical
  • Outcome of meiosis is four (genetically unique) haploid cells
  • Outcome of mitosis is two (genetically identical) diploid cells
20
Q

Homologous chromosomes vs Sister chromatids

A

Homologous chromosomes are chromosome pairs, one from each parent, that are similar in length, gene position and centromere location
Sister chromatids are two identical copies of a single
chromosome that are connected by a centromere.

21
Q

Crossover (HR) occurs at

A

metaphase of meiosis 1

Recombination can generate chromosome crossovers

22
Q

Recombination during meiosis

Process differs slightly from Homologous Repair

A
  • Begins with enzymes Spo11 and Mre11 creating a ‘double stranded break’ in one of the homologous chromosome
  • Nucleotides cleaved from the 5’ ends either side of the break
  • RecA/Rad51 initiates strand invasion
  • Second strand from nicked chromosome ‘captured’
  • DNA polymerase and ligase synthesise new DNA and seal the gaps
  • Results in a double Holliday junction structure
  • Branch migration extends the cross over region or region of heteroduplicity
  • Endonucleases cleave strands to resolve junctions
23
Q

meiosis vs DNA repair

breaking double strand

A

In repair, this may begin as a nick, collapsing the replication fork
• During meiosis, enzymes Spo11 and Mre11 create a break

Double stranded break either spontaneous or induced in one of the homologous duplexes
5’ ends of break processed leaving 3’ overhangs
by Mre 11

24
Q

overhang invasion - recombination

A
  • One of the two 3’ overhangs invades the undamaged duplex at the point where the sequence is complementary
    RecA in Prokaryotes
    Rad51 in Eukaryotes
    RacA like protein catalyzes strand exchange
  • DNA synthesised from the 3’ overhang, using the complementary strand replacing the gap from the break
    the next events
    A: Second strand captured:
    double Holliday junction formed
    B: Invading strand released
25
Q

Holliday model - Invading strand release

A 
Broken double helix re-forms
• DNA polymerase fills any remaining gaps
• Ligation
• No crossover
• This mechanism used for repair
26
Q

Second Strand Capture

A

Additional DNA synthesised to fill gap
• DNA ligated back together
• 3’ end ligated back to the 5’ end of it’s original strand
• BUT with a region now complementary paired to the homologous duplex
• Double Holliday junction

27
Q

double Holliday Junction Model

A
  • Points where single strands separate from double helix: Branch Points
  • Points where strands crossover and join 4 DNA strands: Holliday junction
  • Branch points can move along the DNA strand: branch migration
28
Q

Branch Migration

A
  • An unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point
  • Increasing the regions of cross over/DNA exchange and heteroduplicity
  • Specialised proteins and ATP hydrolysis ensure the branch moves in one direction
29
Q

Programmed Double-Stranded Breaks occur in:

Select one:

a. Double Stranded Break Repair
b. Non-Homologous End Joining
c. Nucleotide Excision Repair
d. Meiotic Homologous Recombination
e. Base Excision Repair

A

This process occurs during meiosis. Spo11 breaks both strands of DNA in one of the recombining chromosomes and works with Mre11 to degrade the 5’ ends, exposing single stranded DNA.

30
Q

Resolution of Holliday junctions

A

• Depending on how nucleases cut DNA, different
product can arise, with or without crossover
• Crossover: exchange of chromatid segments
• Important to be able to identify crossover in the
products
vertical resolution: crossover
horizontal resolution : non-cross over less common

31
Q

Recombination definition:

A

The natural formation in offspring of genetic combinations not present in parents, by the
processes of crossing over of homologous sequences
• Repair-like event preferentially between maternal/paternal
chromosome homologues
• Acts to ensure no two daughter cells are identical nor identical to
parent

32
Q

Recombination can lead to gene conversion

A

Heteroduplexes resulting from mismatched bases following recombination
Detected by DNA repair systems
• Cannot tell paternal/maternal apart
• Random choice of template strand
• Results in ‘conversion’ of one allele to another
• Typically limited to small sections of DNA (part of genes)

BMS1062- (28 decks)

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