**1.1 DNA double-strand break repair**

There are three main repair pathways to deal with DNA double-strand breaks (DSBs) in eukaryotic cells. These include classic nonhomologous end-joining (C-NHEJ) (**Figure 1A**) that modifies and allows for ligation of ends, alternative end-joining (Alt-EJ) that generates short overhangs or exposes small regions of homology via resection to promote ligation of ends (**Figure 1B**), and homologous recombination (HR) that uses a homologous sister chromatid, chromosome, or other sequence as a template to direct repair synthesis (**Figure 1C**) [10, 30]. HR is the most accurate using a homologous template as a donor sequence. DSBs are recombination initiators in both meiotic and mitotic cells [31–33]. However, HR has the most protein involvement, is tightly regulated, largely limited to S phase, and kinetically slow. C-NHEJ is utilized throughout the cell cycle and is kinetically fast. Alt-EJ is less well characterized than the other two and considered a backup repair mechanism when HR or C-NHEJ cannot be used. For a DSB to be repaired by HR or either of the end-joining pathways, damage must first be sensed, then signal transduction pathways must be activated for the DNA damage response (DDR) to bring proteins necessary for repair to the site(s) of damage. Indirect signaling and direct repair protein levels along with histone modifications appear to direct DSB repair pathway selection [34–37]. Despite decades of extensive study of DSB repair, scientists continue to identify and characterize new factors mechanistically involved in DSB end processing, repair itself, as well as pathway choice [38].

### **1.2 End-joining pathway choice**

Repair of DNA DSBs by C-NHEJ or Alt-EJ is characterized by ligation of two DSB ends in close proximity to each other (**Figure 1A** and **B**). Initial binding of the Ku70–80 heterodimer competes with poly(ADP-ribose) polymerase 1 (PARP1) for binding to the DSB. If Ku70–80 binds first there is minimal end processing and C-NHEJ is used [39–44]. For C-NHEJ, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited to the Ku complex. DNA-PKcs can determine if the ends are blunt, as from a nuclease cleavage or from RAG during V(D)J recombination, or if there are overhangs or protein/group adducts. If the break is clean, DNA-PKcs recruits XRCC4-XLF and LigaseIV, and these proteins work together to ligate the DNA ends [39, 42, 45]. However, if there is an overhang or proteins are attached to the break site, DNA-PKcs recruits the ARTEMIS complex for processing.

**77**

**Figure 1.**

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural…*

ARTEMIS can release protein groups and with its nuclease activity to digest the DSB

If PARP1 binds to the DSB before Ku70–80, it immediately adds branched poly(ADP-ribose) (PAR) groups to itself and histones in close proximity. The branched PAR recruit the Mre11-Rad50-Nbs1 (MRN) complex to process the ends and proceed by HR or Alt-EJ. Alt-EJ seems to act as a salvage repair mechanism for when HR and C-NHEJ are blocked. It is likely that Alt-EJ occurs when processing for HR has started following PARP1 binding to the break first, Ku70–80 is depleted, because the DSB ends have proteins bound to block template invasion, or the cell is in G1 phase of the cell cycle no homologous template is readily available for repair. Alt-EJ involves MRN and CtIP to resect the DSB ends in a 3′ to 5′ fashion, termed short range end resection, of 5–25 nucleotides to create short DNA overhangs with small regions of homology. Polymerase θ is utilized in Alt-EJ. After processing, XRCC1 and LigaseIII act in a complex to ligate the ends and remove the overhanging bases. Alt-EJ is more mutagenic than HR or C-NHEJ and associated with chromo-

*The DNA double-strand breaks (DSB) are repaired by the three pathways; these are – A) non-homologous end joining (C-NHEJ) which modifies the ends and allow ligation of the broken ends to repair the DSB; B) alternate end-joining (alt-EJ of EJ) creates short DNA overhangs with small regions of homology and ligates the resected broken ends; and C) homologous recombination (HR) that uses a homologous sequence from sister* 

ends until they are blunt to facilitate ligation of the ends [46].

*chromatid or homologous chromosome or a homologous sequence within the genome.*

somal rearrangements and translocations [44, 47–49].

*DOI: http://dx.doi.org/10.5772/intechopen.95453*

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural… DOI: http://dx.doi.org/10.5772/intechopen.95453*

#### **Figure 1.**

*DNA - Damages and Repair Mechanisms*

implications for genome stability.

**1.1 DNA double-strand break repair**

**1.2 End-joining pathway choice**

and human disease.

predominant use of end-joining [13]. The majority of studies generate targeted DSBs by endonucleases or lasers, and introduce artificial repair substrates into the system [15]. However, exposure to natural compounds can lead to multiple DSBs in a variety of chromatin regions and contexts [16–20]. Understanding how cells respond to these compounds and repair damage caused by them has important

Bioflavonoids are natural compounds in soy, fruits, vegetables, tea, coffee, and wine, and contained in energy drinks and dietary supplements [21–24]. Bioflavonoids are also in pesticides and flame retardants [25–27]. Bioflavonoids inhibit the enzyme topoisomerase II (Top2) to promote DSBs, and recent studies have elucidated the cellular mechanisms used to repair the DSBs induced by bioflavonoids [16, 28, 29]. This chapter will discuss cell type differences in expression and activity of proteins in major DNA repair pathways, summarize findings of cellular response to bioflavonoids and Top2 inhibition, discuss how these compounds trigger alternative end-joining, and conclude with implications for genome instability

There are three main repair pathways to deal with DNA double-strand breaks (DSBs) in eukaryotic cells. These include classic nonhomologous end-joining (C-NHEJ) (**Figure 1A**) that modifies and allows for ligation of ends, alternative end-joining (Alt-EJ) that generates short overhangs or exposes small regions of homology via resection to promote ligation of ends (**Figure 1B**), and homologous recombination (HR) that uses a homologous sister chromatid, chromosome, or other sequence as a template to direct repair synthesis (**Figure 1C**) [10, 30]. HR is the most accurate using a homologous template as a donor sequence. DSBs are recombination initiators in both meiotic and mitotic cells [31–33]. However, HR has the most protein involvement, is tightly regulated, largely limited to S phase, and kinetically slow. C-NHEJ is utilized throughout the cell cycle and is kinetically fast. Alt-EJ is less well characterized than the other two and considered a backup repair mechanism when HR or C-NHEJ cannot be used. For a DSB to be repaired by HR or either of the end-joining pathways, damage must first be sensed, then signal transduction pathways must be activated for the DNA damage response (DDR) to bring proteins necessary for repair to the site(s) of damage. Indirect signaling and direct repair protein levels along with histone modifications appear to direct DSB repair pathway selection [34–37]. Despite decades of extensive study of DSB repair, scientists continue to identify and characterize new factors mechanistically involved in DSB end processing, repair itself, as well as pathway choice [38].

Repair of DNA DSBs by C-NHEJ or Alt-EJ is characterized by ligation of two DSB ends in close proximity to each other (**Figure 1A** and **B**). Initial binding of the Ku70–80 heterodimer competes with poly(ADP-ribose) polymerase 1 (PARP1) for binding to the DSB. If Ku70–80 binds first there is minimal end processing and C-NHEJ is used [39–44]. For C-NHEJ, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited to the Ku complex. DNA-PKcs can determine if the ends are blunt, as from a nuclease cleavage or from RAG during V(D)J recombination, or if there are overhangs or protein/group adducts. If the break is clean, DNA-PKcs recruits XRCC4-XLF and LigaseIV, and these proteins work together to ligate the DNA ends [39, 42, 45]. However, if there is an overhang or proteins are attached to the break site, DNA-PKcs recruits the ARTEMIS complex for processing.

**76**

*The DNA double-strand breaks (DSB) are repaired by the three pathways; these are – A) non-homologous end joining (C-NHEJ) which modifies the ends and allow ligation of the broken ends to repair the DSB; B) alternate end-joining (alt-EJ of EJ) creates short DNA overhangs with small regions of homology and ligates the resected broken ends; and C) homologous recombination (HR) that uses a homologous sequence from sister chromatid or homologous chromosome or a homologous sequence within the genome.*

ARTEMIS can release protein groups and with its nuclease activity to digest the DSB ends until they are blunt to facilitate ligation of the ends [46].

If PARP1 binds to the DSB before Ku70–80, it immediately adds branched poly(ADP-ribose) (PAR) groups to itself and histones in close proximity. The branched PAR recruit the Mre11-Rad50-Nbs1 (MRN) complex to process the ends and proceed by HR or Alt-EJ. Alt-EJ seems to act as a salvage repair mechanism for when HR and C-NHEJ are blocked. It is likely that Alt-EJ occurs when processing for HR has started following PARP1 binding to the break first, Ku70–80 is depleted, because the DSB ends have proteins bound to block template invasion, or the cell is in G1 phase of the cell cycle no homologous template is readily available for repair. Alt-EJ involves MRN and CtIP to resect the DSB ends in a 3′ to 5′ fashion, termed short range end resection, of 5–25 nucleotides to create short DNA overhangs with small regions of homology. Polymerase θ is utilized in Alt-EJ. After processing, XRCC1 and LigaseIII act in a complex to ligate the ends and remove the overhanging bases. Alt-EJ is more mutagenic than HR or C-NHEJ and associated with chromosomal rearrangements and translocations [44, 47–49].

#### **1.3 Homologous recombination requires chromatin remodeling and DDR**

To initiate HR (**Figure 1C**), PARP1 is recruited to the DSB first and immediately adds branched PAR groups to itself and histones in close proximity. The branched PAR recruit the MRN complex and inactive ATM kinase dimers with the acetyltransferase TIP60 attached. PARG quickly removes the PAR groups allowing the MRN complex to bind to the DSB. MRN allows ATM to bind at the DSB and activate through auto-phosphorylation and acetylation by TIP60, thereby allowing TIP60 to dissociate. Once active, ATM will phosphorylate a large number of target proteins including the MRN complex and CtIP that process DSB ends [12, 34, 42, 50].

Chromatin remodeling is extensive and required for HR-mediated DSB repair. Histone H2AX is phosphorylated by ATM as well as acetylated by TIP60. Phospho-H2AX (γ-H2AX) has some chromatin remodeling functions and acts as a signal to recruit additional proteins involved. γ-H2AX will spread away from the DSB to decorate chromatin up to 2 Mb away. MDC1, which assists with chromatin remodeling, becomes phosphorylated by ATM and recruits RNF6 dimers that have ubiquitination functions. HERC2 associates with phosphorylated RNF6 and appears to recruit PIAS4 which has SUMOlyation capabilities. RNF6 becomes SUMOlyated and mono-ubiquitinates histones in the area, which recruits RNF168, another ubiquitin ligase, that is SUMOlyated and poly-ubiquitinates nearby histones. The poly-ubiquitin trees tether BRCA1-A complexes by RAP80 mediators. These complexes cause histone modifications that bring in 53BP1, which has more histone remodeling functions and can inhibit MRN and CtIP-mediated end resection [34, 39, 43, 50].

Phosphorylation of target proteins by ATM also triggers DDR. Chk2 has protein kinase activity allowing it to phosphorylate a number of effector proteins in the cell cycle checkpoint including p53 which can be modified by either Chk2 or ATM (or ATR or Chk1). ARF protein (p14) seems to stabilize TIP60 interactions with ATM for better activation and is associated with maintaining genome stability [34].

While the histone remodeling is occurring and other proteins are being recruited, MRN and CtIP resect the DSB ends short range end resection, then Exo1 or Dna2 nucleases act in long range end bidirectional resection in a 5′ to 3′ direction away from the DSB. Exo1 has dsDNA nuclease function, while Dna2 must act with a helicase like BLM or WRN to unwind DNA for its ssDNA nuclease abilities [34, 37, 43, 51]. While long range end resection is occurring, RPA binds to the 3' ssDNA overhang to protect from nucleases. After this resection, one type of HR can occur called single strand annealing (SSA), where the two pieces of RPA coated DNA associate with one another with the help of Rad52 and if regions of homology are found they anneal to one another. Non-homologous flaps are cleaved off by enzymes like XPF-ERCC1 and ligated by LigaseIII. This type of HR can cause large deletions [12, 43, 50, 52].

Canonical HR, as well as break-induced replication (BIR) and synthesis-dependent strand annealing (SDSA) use BRCA 1 and 2 with Rad51 for homology searches that cause strand-invasion, D-loop formation and resolution/dissolution. RPA must be dissociated from the ssDNA for Rad51 binding, mediated by DSS1 and BRCA2 which displace RPA and stabilize ATP on Rad51 increasing its binding affinity for the ssDNA. Once Rad51 is loaded on the DNA and the nucleofilament has formed, it can invade neighboring DNA to search for homology with BRCA1 [34, 43, 50]. Homology less than 7 nt in length is a weak interaction and Rad51 not sufficient to initiate HR, but 7 nt or longer allows the strand to interact more strongly [50]. If significant homology is present, the ATP on Rad51 is hydrolyzed causing the dsDNA to dissociate and the nucleofilament anneals with the template strand.

**79**

**Figure 2.**

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural…*

RPA stabilizes this D-loop formation by binding to the displaced strand. DNA Polymerase δ or ε uses the invading strand as a primer to initiate synthesis [12, 39, 50, 53]. Resolution can happen with crossover or non-crossover products and different sets of resolvases mediate this process. For one-sided ends that utilize BIR, DNA Pol δ is used and synthesis continues until the end of the chromosome causing gene

Topisomerase II (Top2) is a regulatory enzyme that relaxes supercoiled DNA for transcription (Top2β) and replication (Top2α). As shown in **Figure 2**, Top2 acts in a multistep cleavage and religation reaction: (1) Top2 binds to two dsDNA molecules at Top2 recognition sequences; (2) a transient DSB is generated in the first DNA helix (G-segment) creating a cleavage complex; (3) ATP hydrolysis drives a conformational change allowing the second dsDNA helix to pass through the DSB; (4) Top2 mediates religation of the DSB and the T DNA segment is released; (5) the G DNA segment is released and the enzyme returns to its original conformation (**Figure 2**). A catalytic Top2 inhibitor such as dexrazoxane acts to prevent DNA from binding to Top2 at step 1 preventing any part of the catalytic cycle [55–59].

*Top2 acts in a multistep cleavage and religation reaction. 1) Top2 binds the G and T dsDNA molecules at Top2 recognition sequences. 2) ATP binding catalyzes the DNA DSB in the G segment, which allows the T segment to pass through the break. (3) ATP hydrolysis drives a conformational change allowing the second dsDNA helix to pass through the DSB; (4) Top2 mediates religation of the DSB and the T DNA segment is released; (5) the G* 

*DNA segment is released and the enzyme returns to its original conformation.*

*DOI: http://dx.doi.org/10.5772/intechopen.95453*

conversion that can be highly mutagenic [54].

**2. Topoisomerase II, inhibitors and poisons**

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural… DOI: http://dx.doi.org/10.5772/intechopen.95453*

RPA stabilizes this D-loop formation by binding to the displaced strand. DNA Polymerase δ or ε uses the invading strand as a primer to initiate synthesis [12, 39, 50, 53]. Resolution can happen with crossover or non-crossover products and different sets of resolvases mediate this process. For one-sided ends that utilize BIR, DNA Pol δ is used and synthesis continues until the end of the chromosome causing gene conversion that can be highly mutagenic [54].
