170525 WOORI BAE DNA damage types and repair

170525 WOORI BAE

DNA damage types and repair pathways Methylating agent MMS: methyl methanosulfate MNNG: N-methyl-N’-nitro-N-nitroguanidine 8 -oxo-G: 8 -oxoguanine Hoeijmakers, 2001 Mispaired bases are replaced by the correct bases through the mismatch repair (MMR) mechanism, whereas chemical alterations of bases lead to their replacement by a process called base excision repair (BER).

① ② AP site Base Excision Repair (BER) ③ BER consists of two main pathways, ⑤ ④ 3’-OH, 5’-d. RP l short patch repair (SN-BER) l long patch repair (LP-BER) The first step of BER involves recognition, base removal and incision. The choice between short-patch BER or long-patch BER depends on the state of the 5’ deoxyribose phosphate (5’d. RP) terminus. In the final step ligation is performed. 2003) (Christmann et al.

Simplified overview of the four pathways repairing DSBs in higher eukaryotes After the generation of a DSB, PARP-1 is one of the first molecules to arrive at the free DNA ends. This may lead to the recruitment of the MRN-complex, which carries out initial end resection in all cell-cycle phases. Krejci et al. , 2012

* Role of poly(ADP-ribose) polymerase 1 (PARP-1): nick sensor !! After base excision and AP-site incision, poly(ADP-ribose) polymerase 1 (PARP 1) may play an enzymatic role at the resulting 1 -nucleotide (nt) gap, possibly by inducing a relaxed chromatin structure and allowing access of repair enzymes to the DNA and/ or by coordinating the actions of downstream BER events (Dantzer et al, 1999; Prasad et al, 2001; Le Page et al, 2003). Genomics Proteomics Bioinformatics 14 (2016) 131– 139

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Experimental strategy of the plaque-based assay for detecting patch formation during BER Plasmid based assay combinations of appropriate restriction enzymes; One enzyme was utilized to probe for repair at the damage site, and one enzyme was utilized to probe for patch formation. § percentage of repair events that occur via 5’ and 3’ patch formation can be quantified Completely repaired DNA will be linearized by restriction digestion and will not form plaques, while unrepaired DNA or products of incomplete BER will be resistant to digestion, remain circular, and form plaques ** εA: representative BER substrate DNA adduct, ethenoadenine

Fig 1. A new sub-pathway of LP-BER in live cells involves 5’ patch formation εA repair patch mapping BER 5’ patch formation hypothesis Patch formation size Total 18 -20 nt RECQ 1 ERCC 1 XPF LP-BER as the mechanism for εA repair in-cell repair of εA over 24 h generated a patch size of 12 bases 3’ to the lesion as well as 7– 9 bases 5’ to the lesion newly!! *traditional LP-BER patch size: 2 -12 nt the involvement of a 3’– 5’ helicase at the incised AP site and an associated 3’ flap endonuclease

Experimental timeline for plaque-based repair assay in the presence of gene-specific si. RNA. To probe for the role of RECQ 1 in BER, In RECQ 1 KD HCT 116 cells, § § only 10% of εA was repaired in 24 h both 5’ and 3’ patch formations were abolished RECQ 1 is required for total repair and patch synthesis during 5’ gap-mediated LP-BER Suppl. RECQ 1 si. RNA did not affect the expression of other Rec. Q family helicases, indicating that RECQ 1 is specifically needed for 5’ patch formation since other available Rec. Q helicases could not rescue the 5’ patch formation in the absence of RECQ 1

Fig 1. RECQ 1, PARP 1, RPA, and ERCC 1 -XPF are necessary for 5’ patch formation and BER RPA was essential for BER of εA ERCC 1 -XPF was essential for BER

Fig 2. 5’ repair patch in BER is created by the formation of a 5’ gap The linear M 13 mp 18 plasmid DNA and the expected sizes and results for digestion * * * RECQ 1 was not involved in 3’ patch formation during BER using a similar strategy with εA at the Pst. I site 5’ gap formation did not occur BER employs a mechanism that includes RECQ 1 dependent formation of a gap on the 5’ side of the lesion

5’ gap formation elimination of any one of the six proteins resulted in abolition of the gap formation To identify the enzymes essential to the formation of the 5’ gap, in vitro gap formation assay with purified proteins, including MPG, APE 1, PARP 1, RECQ 1, ERCC 1 -XPF, and RPA all six of these proteins are required for 5’ gap formation during repair

Fig 3. 5’ gap formation during BER is modulated by PARP 1 -RECQ 1 relationship To measure the exact size of the 5’ gap, The BER 5’ gap is 9 nts in length The 8 nts 5’ to the lesion combined with the 12 nts 3’ (Fig 1 B) to the lesion results in a total patch size of precisely 20 nts in the newly discovered 5’ gap-mediated LP -BER Original lesion 1 nt included, reactions containing all six proteins resulted in a 5’ gap that was precisely 9 nt in length (including the original lesion site) the formation of the 5’ gap occurs post-base excision and AP-site incision, and the single-nucleotide gap (1 -nt gap) is the critical DNA substrate for the other four proteins (PARP 1, RECQ 1, RPA, and ERCC 1 -XPF) to form the 5’ gap

RECQ 1 may be acting as a biological PARP 1 inhibitor, and this inhibition may be critical for the gap formation initiated by RECQ 1 in BER gap formation - inhibited by addition of higher concentrations of PARP 1’s substrate for PARylation, NAD+(≥ 200 μM). - did occur in the presence of physiologically relevant concentrations of NAD+, which is found at a concentration of ~100 μM in the nucleus and cytoplasm (Koch-Nolte et al, 2011). gap formation was not observed § PARP 1, RPA, and RECQ 1 were recruited to the 1 -nt-gapped substrate independently PARP 1 auto. PARylation induced by 1 -nt-gapped DNA was significantly inhibited by the presence of RECQ 1 with 5 μM NAD+ PARP auto. PARylation was unaffected by the presence of RECQ 1 under excess NAD+ § RECQ 1 recruitment to the substrate was significantly increased in the presence of PARP 1 or RPA § the 5’ gap formation step in LPBER revealed the potential importance of the relationship between RECQ 1, PARP 1, and RPA in regulating BER

Fig 4. l PARP activity-mediated SN-BER is the mechanism of repair in the absence of RECQ 1 l RECQ 1 -PARP 1 regulation of BER in genomic DNA RECQ 1 KD should allow PARP 1 PARylation and activation of the SN-BER pathway since it is known that PARP 1 PARylation coordinates SN-BER (Caldecott et al, 1996; Dantzer et al, 1999) the role of PARP activity in the RECQ 1 independent repair pathway with the addition of olaparib, a small-molecule PARP inhibitor (PARPi), to the plaquebased repair assay

§ the RECQ 1 - independent, slower mechanism of BER was abrogated in the presence of olaparib in HCT 116 § The RECQ 1 -independent BER mechanism was also abrogated in POLβ-deficient cells § RECQ 1 -mediated BER is indeed the major mechanism of BER § PARP activity-mediated repair was occurring primarily in the absence of RECQ 1 § the RECQ 1 KD cells were more sensitive than the control cells to alkylating agent in combination with PARPi, which is consistent with the absence of both BER sub-pathways

Fig 5. 5’ gap formation-mediated LP-BER is relevant for repair of oxidative DNA damage real-time PCR-based repair assay Representative BER substrate DNA adducts, 8 -oxoguanine (8 -oxo. G) and 5 -hydroxyuracil (5 -OHU), accumulated which are repaired by bifunctional DNA glycosylases. 8 -oxo. G is recognized and excised by 8 -oxoguanine DNA glycosylase (OGG 1) while 5 -OHU is recognized by nth-like DNA glycosylase 1 (NTH 1) and Nei family DNA glycosylases RECQ 1 KD HCT 116 cells, PARP activity-mediated repair of oxidative DNA damage was occurring in the absence of RECQ 1 § § The repair of both adducts occurred via 5’ gap formation dependent on RECQ 1 no SSBs

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