Repair of DNA Double-Strand Breaks - Radiation Damage in Biomolecular Systems - page 332

DSB sensors, a co-activator of DSB checkpoint signaling ( e.g ., by activating ATM),
and as an effector protein both in the NHEJ and HR [ 46 - 50 ]. In addition, multiple
regulatory feedback loops complicate our understanding of the temporal order of
protein-protein and protein-DNA interactions involved in DDR. For instance, the
identity of the first sensor necessary to recognize DSB lesions is in dispute.

20.3 “Biochemistry” of DSB repair pathways:

What we think we know

20.3.1 Non-homologous end-joining (NHEJ)

AT M k i n a s e [ 51 ], Ku proteins, and MRN complex [ 47 - 50 ] are among the candidates
to be the “initiators” of DDR. ATM kinase is evidently the capellmeister, with
a central role in regulating both the NHEJ and HR repair pathways, cell cycle
checkpoints in G 1 =

M, apoptosis and induction of specific
transcriptional programmes in response to DSB [reviewed in 52 - 54 ]. Within
intact cells, ATM exists as an inactive dimer. After DSB formation, ATM is
autophosphorylated and dissociates into active monomers [ 55 ], which also results
in the release of protein phosphatase 2A from ATM, so the activation of ATM is
not further inhibited [ 56 ]. Full activation and amplification of the ATM signal is
stimulated by MRN complex and DNA-PK bound to the sites of DSB [ 57 , 58 ],
where “semi-activated” ATM interacts with a broad spectrum of proteins. One
of the ATM targets is the MRN complex that in a feedback loop activates ATM
and enables its additional accumulation at the sites of DSB. The MRN complex
is therefore another important and versatile protein that potentially participates in
DSB recognition and initiation of DSB repair [ 12 , 50 ]. It has multiple additional
roles in both NHEJ and HR, especially in DSB-end processing [ 59 - 62 ], spatial
stabilization of ends [ 63 , 64 ], and cellular signaling [ 50 ]. One of the first events in the
NHEJ pathway is also binding of Ku heterodimer (Ku70/Ku80) to free DNA ends,
where it stabilizes damaged chromatin, prevents DNA ends resection and potentially
recognizes the lesion [ 65 ]. The most probable scenario therefore includes several
parallel processes participating in sensing DSB, DDR activation, and signaling, with
mutually stimulating effects.
Despite the complicated initiation step outlined, recent results [ 66 - 78 ]showed
that most DSBs (about 85%, see Sect. 20.3.4 , 20.4.4 ) can be repaired in principle
with the participation of only five “core” proteins including Ku heterodimer, DNA-
PKcs, XRCC4, and DNA ligase 3. Therefore, the NHEJ pathway per se (absent
cellular signaling) may begin by the binding of Ku proteins to DSB ends, which
enables their interaction with DNA-PK catalytic subunit (DNA-PKcs, a member of
PIKK subfamily of PI-3 kinases [ 72 ]) and its activation [ 73 , 74 ]. Once active, DNA-
PK holoenzyme phosphorylates several targets at the site of the DSB, including
itself, which results in the dissociation of DNA-PKcs subunits from DSBs [ 75 ]and
their exchange for NHEJ or HR proteins. DNA-PK (and Ku) thus probably tethers

S, intra-S and G 2 =