DNA repair pathway (64-66). For the purpose of

DNA damaging
agents such as ionizing radiation, topoisomerase II inhibitors, antitumor
antibiotics, such as bleomycin, and cellular recombination processes can induce
DSBs directly (61), while other agents, such as ultraviolet
(UV) light, and oxidative free radicals can cause DSBs when the replication
fork encounters unrepaired SSB (62). DSBs pose a major threat to cell survival
and, unless repaired, lead to chromosomal rearrangements (translocations or
deletions) or cell death. These detrimental effects help accelerate aging and
promote carcinogenesis (63). There are three main DSBR pathways
homologous recombination (HR), NHEJ and alternative NHEJ (alt-NHEJ) pathway.
HR, an error free repair pathway, requires the homologous strand at the time of
repair to use as a template and is active during DNA replication (S phase) and
G2 phase (64-66). On the other hand, NHEJ functions in all
phases of the cell cycle and does not require homology between DNA strands, and
is considered an error prone repair pathway (64-66). For the purpose of this thesis, I will only
focus on NHEJ and alt-NHEJ because PNKP participates in DNA end processing in
these pathways (67). Nonhomologous end-joining (NHEJ):
classic and alternative pathways

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!

order now

NHEJ is a
major mechanism of DSBR (68). The Ku complex, a heterodimer of two
protein subunits Ku70 and Ku80, acts as a sensor for DSBs. The presence of Ku
complex at the DNA ends of DSB is essential for subsequent DNA end processing
and ligation (69, 70). DNA-dependent protein kinase catalytic
subunit (DNA-PKcs) is recruited to Ku at DNA ends of DSB, becomes activated and
catalyzes synapsis of the DNA ends by dimerization (69, 70). 
Another core complex, XRCC4-lig4-XRCC4 like factor (XLF), is also
recruited to sites of damage. XRCC4-lig4-XLF induces DNA-PKcs
autophosphorylation, dissociation, translocation of the Ku heterodimer along
the DNA away from the ends, and recruitment of end processing factors (70). A number of end processing enzymes and
polymerases are recruited to the site of damage by interacting with the core
complex (62,
70, 71). PNKP is one of the key enzymes required for the processing of
radiation-induced DSBs.  PNKP is
recruited to the site of damage by interacting with phosphorylated XRCC4 via
the PNKP FHA domain (72). 
Ligation of DNA ends is catalyzed by lig4. Finally, the Ku complex is
removed from DNA ends by proteasomal degradation mediated by ubiquitination by
Cullin and RNF8 (70). Figure 1.3 shows a model depicting the
steps involved in the conventional NHEJ pathway.

A substitute
of classical NHEJ, alt-NHEJ, is available when the cell is deficient in NHEJ
components and relies on microhomology (MH), the tendency of the two DNA ends
to align themselves at short regions of homology within the two ends (73-75). The main attributes of alt-NHEJ are large
deletions, insertions and a high frequency of chromosomal translocations (76). The damage is detected in this situation by
PARP-1, which facilitates the recruitment of the MRN complex to bridge the DNA
ends, following the loading of CtlP to facilitate MH searches (77-79). The ligation process is mainly driven by
the activity of the XRCC1/lig3 complex and on some occasions lig1, where MH is
not required (80). In recent studies, it was shown that
polymerase ? (Pol ?) performs terminal transferase activity at the 3?-termini of resected DSBs to mediate MH (81,
82). PNKP is also involved in this process and is likely partially
regulated by IR-induced phosphorylation of PNKP
by Ataxia telangiectasia mutated (ATM) and DNA-PKcs (83, 84). An in vivo study showed that the inactivation of DNA-PKcs
and/or ATM led to reduced PNKP levels at DNA damage sites (84). Based on the
observation that depletion of PNKP does not alter the level of sister chromatid
exchanges, it was surmised that PNKP does not participate in HR, the other
major DSB repair pathway (85).


I'm Mary!

Would you like to get a custom essay? How about receiving a customized one?

Check it out