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

*DNA - Damages and Repair Mechanisms*

[34, 39, 43, 50].

deletions [12, 43, 50, 52].

**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

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

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.

**78**

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].

#### **Figure 2.**

*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.*
