NHEJ

the cell decision to repair though one pathway or the other is taken at the ends → the DSB can be processed differently triggering different pathways for the repair

2 general pathways for DSB:

  • NHEJ
  • HDR

mechanism to repair DSB derived form the studies of human cells hypersensitive to mutations → associated to severe immunodeficiency

the patients defective in NHEJ are sensitive to IR (that cause DSB so chromosomes rearrange in strange ways) and severely immunodeficient (they have mutations in genes involved in the maturation of the lymphocyte)

List of NHEJ genes :


  • XRCC6 (in mice)
  • KU70 (in humans)
  • DNA-PK
  • LIG4

studying immunodeficiency + functionality of those genes because of radiation sensitivity

➜ IgH is fusing with c-myc
locus IgH on chr 14 is sometimes rearranged because of a translocation involving chr 8

typical of Burkitt lymphoma (BI)

one part of the chromosome translocates to the other chromosome leading to a reciprocal translocation

this translocation happens also with other chromosomes, not just with chr 8
➜ the particular locus encoding for IgH is a hotspot for recombination since this locus is exposed to programmed DSB formation


this locus is the VDJ locus

NHEJ is involved in the repair of the DSB created during the VDJ recombination and CSR in lymphocytes maturation
if we have mutations in the NHEJ factors → prevent Ig genes maturation leading to severe immunodeficiency

Chr 14: IgH locus: cassettes formed by different repeats
→ V segments
→ D segments
→ J segments
→ constant region exon

V segments are rearranged in a way in which just one cassette is joined to another cassette + the variable regions exons.


The gene is then transcribed and the typical Y structure the antibodies is formed

the tip part is variable → many types of antibodies

high variability ➜ reached by the rearrangement of these segments in different ways but happens in very specific steps
➜ D is joined to J and then this part is joined to the V part

VDJ RECOMBINATION

RAG nucleases

cut specific consensus sites present on the cassettes leading to the cut of the chromosomes with the formation of ends
→ these ends are processed because they contain a hairpin structure.
After the cut we will have clean ends that can be joined (one of the steps in gene maturation)

Now we know that this process involves DNA-PK and the KU complex as well as the LIG4

after the VDJ if formed it is then joined to the constant region
the presence of one C part or another determines if

  • the antibody is localized in the membrane
  • is is secreted in the body
  • it is secreted outside on the surface

with Epistasis analysis

rad52 is involved in HR while KU in NHEJ

by inducing radiations in the double mutant of KU and rad52 we see that the sensitivity increases compared to the one of single mutants, so we can say that KU and rad52 are involved in 2 different pathways responding to DSB

KU COMPLEX

Heterodimer→ KU70 + KU80

associated at the ends of the DSB

when deposed, with DNA-PK, creates a platform though which other enzymes like ligases, nucleases and polymerases could be associated

we have to distinguish

c-NHEJ

alt-NHEJ

after the DSB there are the 2 parts that have to be joined.

Sometimes the joining of the 2 parts could lead to some imprecise joining because if there is no homology between the two parts a few bases have to be removed or added to the break point

NHEJ allows to reseal a break but it is not always very accurate

error - prone

phases

1) KU heterodimer (acts as a docking station for loading other NHEJ factors)
2) DNA-PK
3) Polymerases
4) XRCC4, XLF and LIG4
5) PAXX

precise: when blunt and complementary ends are present
imprecise: when ends are less compatible (they can be altered) and in this case nucleases are needed to delete or insert bases

the imprecise case, the more frequent, exposes the cell to accumulating variations at the break point

worse situation ➜ when there are 2 DSB in 2 different chromosomes

there could be erroneous repair,

  • and in this case 2 different loci are altered
  • one arm of the chromosome can be resealed with another arm of the chromosome → formation of an acentric chr and a diacentric chr ( cells die usually)
  • reciprocal translocation (as in Bl)
  • formation of one-ended DSB during replication

if KU is not present, some nucleases extensively remove bases from both sides leading the the exposition of the strands with few bases perfectly compatible for annealing the nucleases will then remove the flaps and ligase will rejoin the ends

joining can be mediate by microhomologies

Micro-Homology Mediated End Joining (MME)

allows the re-join of the ends thanks to the exposition of a short region of homology on both sides of the break

leads to severe rearrangements since now we will have extensive deletions

the entire part after the resection of the ends, and after the action of the ligase, is lost

completely KU independent

BUT requires PARP polymerase that will create the scaffold structure for the recruitment of the nucleases (MRE11 complex)

LIG4 is also not recruited

they recruit LIG1 and LIG3

chromosome rearrangements

experiment

KU -/-

wt cells (KU present)

no translocation → normally even if inaccurate the joining occurs in cis
rarely can cause translocations since it happens in trans

since KU is not present, the two ends cannot be tied together so they start moving and are exposed to the processes that might end up expose micro-homologies ➜ alt-NHEJ MMEJ
This can lead to cis joining (deletions)or trans joining (translocations)

if we have two breaks in 2 different chromosomes

KU is normally deposited at the end of the break and forms a sort of bridge between the two parts allowing for NHEJ events to occur

KU is an oncosuppressor gene

it maintains the synapsis between the two ends of the DSB suppressing or limiting the events that lead to movement

  • suppressing translocations
  • protecting them from deletions

Philadelphia chromosome

due to a reciprocal translocation between chr 9 and chr 22 → formation of a longer chr and a very small one (Philadelphia)

this causes the fusion of 2 genes : BRC + ABL that lead to leukaemia

the translocation is induced by alt-NHEJ and at the break point there is the annealing between part of the chr22 and chr9.
The two parts then re anneal because of complementarity and the two ends are reapired though MMEJ

special case

SENATAXIN helicase in DSB repair

mutations in this gene cause 2 rare but severe neurological disorders associated with different types of ataxia → AOA2 and ALS4

this helicase is able to separate the filaments of the DNA double helix (as typical) but especially it separates filaments between a DNA and a RNA filaments

➜ involved in the termination of transcription
(→ but also other activities concerning the DNA and the RNA)

involved in DSB repair
⇒ DNA-RNA molecules could be formed during certain repair mechanisms

after the DSB is formed , the cell synthesize a short RNA close to the ends → R-loop formation and sen1 normally removes these transcript leaving clean ends that can be processed either by NHEJ or by HDR

without this helicase the transcript remain there and this may lead to translocations with alt-NHEJ or deletions with in cis c-NHEJ repair

SSA
(Single Strand Annealing)

it is a mechanism that was observed similar to alt-EJ in humans

it is a mechanism depending on the annealing of the strands and leads to the deletion of some sequence

DNA annealing and pairing reactions in recombination

1. Strand pairing: a double strand is formed by the annealing of two complementary strands
2. Pairing + Exchange: we have a circular strand and a double strand with end (one of the strands anneals to the complementary circular one)
3. Strand Invasion: a strand anneals to the target homologous sequence of the double helix
4. Strand Exchange: between double strand DNA ends (a strand of the double helix is exchanged with a strand of the double circular DNA)

all these mechanisms are not spontaneous and are instead stimulated by specific proteins

rad52

a small protein that forms a complex formed by 11 subunits

ring-like structure → the DNA wraps around it

it interacts with ssDNA and allows the annealing and pairing of two strands coming from 2 chrs or from the 2 sides of the break

steps

rad52 mediates the annealing of the 2 DNA strand that is case of homology will anneal

the nuclease will cut the flaps (the ssDNA that are not homologous)

the short gasp are sealed with polymerases and ligases

→ this system allows the restoration of the intact chromosome but at the end it will be shorter since some sequence will be deleted

RESULTS IN SMALL DELETIONS → NOT A CONSERVATIVE REPAIR

The kinetics of this mechanism can be followed

→ HO induction, so break formation
→ Southern blot with probe to see the break
→ Over time we can see the appearance of the bad (break) and how it changes during the repair mechanism
→ the cut band disappears and the the product appears after a while
→ the sequence changes also after the repair

  • the break is formed
  • it is converted to single strand→ 3' end single strand tail
  • this will invade the target sequence though the invasion mechanism
  • DNA replication starts copying the target sequence on the donor sequence

BIR

allows the complete replication of an entire arm of the chromosome until the telomere

this happens though the formation of a replication fork

outcome

the donor chromosome remains untouched while the broken one is completely new.
we will have a part of the chromosome is still the same while the other is completely reconstituted as a result of the copy of the replication

THE REPAIRED CHROMOSOME IS NOW EQUAL IN PART TO THE DONOR CHROMOSOME
➜ THERE IS LOH (loss of heterozygosity), that is one of the robust characteristics of the diploid genome

leads to half-CO or no-CO

SDSA

the invasion and the pairing is rejected after a while and the newly synthesised sequence re-anneal with the broken chromosome

no-CO

dHj

after the invasion on the donor template the synthesis goes on but then the other end on the other side is also captured (second end capture)

two types of joining molecules on the 2 sides → dHj

CO or no-CO

the MRE 11 complex is recruited at the ends from PARP1 and performs the resection