- cell-cycle independent - error-prone - VDJ and class switching

- cell cycle dependent - accurate

- can be (a)symmetric - asymmetry leads to crossing over (bad for mitosis!) - leads to LOH (cancer)

DSB Repair Flashcards by Grace Beglan

Structure

DSB intro
HR
NHEJ

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Describe DSB severity

severe
loss of genetic information/translocations
cell death (apoptosis)

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Causes of DSBs can be

exogenous
endogenous

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Exogenous causes of DSBs

IR
chemotherapeutics

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Endogenous causes of DSBs

meiosis
mitosis
VDJ recombination
class switching
mating-type switching
enzymes
DNA replication

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Describe how DNA replication results in DSBs

replication forks encountering a ssb

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Centromeric DSB repair

specialised sub-pathway (Top2alpha)
necessary due to the catenation of highly repetitive centromeric DNA
failed repair can lead to chromosomal breakages

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Identifying proteins involved in DSB repair:

yeast genetics (Rad)
human genetic syndromes (Artemis, BRCA1)
biochemistry (DNA-PK)

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NHEJ - the basics

cell-cycle independent
error-prone
VDJ and class switching

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cell cycle dependent
accurate

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Types of HR:

1) single strand annealing (leads to deletions)
2) DHJ pathway
3) synthesis-dependent strand annealing pathway

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HR - single strand annealing:

1) MRN, CtIP, Exo1 and DNA2 cause resection (generates ssDNA with a 3’OH overhanging tail)
2) homologous sequence anneals
3) flap removal
4) ligation

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HR - DHJ pathway:

1) MRN, CtIP, Exo1 and DNA2 cause resection (generates ssDNA with a 3’OH overhanging tail)
2) one end invades the sister chromatid
3) 2nd end captured by D loop
4) DHJ cleavage
5) dissolution/resolution

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DHJ cleavage

can result in gene conversion/recombination; either way, the DSB is repaired

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dissolution - the basics

preferred
does not lead to cross-over events

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resolution

can be (a)symmetric
asymmetry leads to crossing over (bad for mitosis!)
leads to LOH (cancer)

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Dissolution - the mechanism

1) DHJs further processed by helicase unwinding
2) HJ branches migrate towards each other
3) catenation
4) DNA topoisomerase 3: decatenation

HR - synthesis-dependent strand-annealing pathway:

MRN, CtIP, Exo1 and DNA2 cause resection (generates ssDNA with a 3’OH overhanging tail)
one end invades sister chromatid
invading strand dissociates and anneals to the other, broken end
polymerisation
ligase action

What does HR synthesis-dependent strand annealing result in?

repair without changing the sister chromatid

HR proteins

1) NHEJ-displacement proteins
2) end-resectors
3) sister chromatid invasion
4) helicase

NHEJ-displacement proteins

MRN moves 3’->5’, displacing Ku70-80
Rad51 “recombinase” deposition on 3’ overhang?

Why is NHEJ important?

predominant repair mechanism in mammalian cells
failure is associated with major translocations
necessary for cancer treatment survival
correlated with immune function

HR and NHEJ collaborate in DSB repair…

double deficient mice are acutely radiation sensitive

C-NHEJ - the basics

evolutionarily conserved
Ku70-80, DNA-PK, Artemis, Pol mu, Lig IV, XRCC4, XLF
perfectly compatible overhangs are repaired error-free
IR-induced lesions are error-prone
VDJ and class switching

C-NHEJ - the mechanism

1) DSB recognition 2) termini processing 3) synthesis 4) ligation

What are the possible C-NHEJ outcomes:

1. direct DNA end-end fusion (no strand exchange or homologous DNA) 2. IR/ROS-induced lesions are refractory; require further processing to prevent damage

Ku70-80 heterodimer

- ~86 - preformed ring: dyad-symmetrical; forms torroid - binds to and encircles cut ends to protect them from degradation - recruits NHEJ proteins (80 C-terminus: DNA-PKcs targeting and activation) - abundant (~400,000 molecules per cell, esp. in nucleus)

Artemis

- abnormal genetics endonuclease (5' -> 3') - interacts with PK-cs - facilitates Lig IV

Polynucleotide kinase (PNK)

transformed LigIV 5' hydroxyl -> phosphate

DNA PK-cs

- Ser/Thr kinase - phosphatidylinositol-3-OH kinase (PI3K)-related kinase (PIKK) family - 469kDa - facilitates LigIV

Lig IV

- ATP-dependent - facilitated by Ku70-80, XRCC4, XLF

A-NHEJ

- aka microhomology-mediated end-joining - if C-NHEJ is inactivated (e.g. in mitosis) - Ku70-80, DNA-PK, Lig IV - independent - error-prone (reciprocal translocation) - not very well understood

A-NHEJ mechanism:

1) MRN cause resection (generates ssDNA with a 3'OH overhanging tail) 2) opposing microhomologies align 3) poly-ADP ribose polymerase (PARP) adds ADP and ribose to either side of the chain 4) recruits DNA Lig III

HR-NHEJ choice

- M: A-NHEJ - G1: NHEJ - S: HR (CtIP)

VDJ recombination

- required in early TCR and Ig production - randomly combines VDJ segments

VDJ mechanism

1) RAG1/2 generate DSBs and RSSs, generating hairpins at DJ ends 2) NHEJ enzymes process to open the hairpins 3) end ligation

RSS

recombination signal sequence

class switching

- changing the constant region of the Ab heavy chain - does not affect antigen specificity - DSBs generated in 2x S regions and intervening sequence excised

Mechanism of class switching

1) AID de-aminates C and S region: U 2) UNG creates an abasic site 3) cleaved by APE1 to create nicks; staggered DSBs 4) NHEJ processes and joins

S region

switch region

UNG

- uracil-DNA glycosylase

APE1

endonuclease