**4. Plants as model organisms to study DNA repair**

Plants and animals share a surprisingly high degree of conservation among their abilities to repair damaged DNA (please also see Table 1 at the end of this passage for an overview of genes involved in DNA repair in the model plant *Arabidopsis thaliana*). While mammalian researchers have very valid and scientifically relevant reasons to use animal subjects, plants

activities of HR and NHEJ (Bakalkin et al., 1994; Subramanian and Griffith, 2005; Gatz and

Activation of p53 after DNA damaging conditions is achieved by phosphorylation by the checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATAXIA TELANGIECTASIA AND RAD3 RELATED (ATR) (Canman et al., 1998; Tibbetts et al., 1999). While recent studies imply that ATM is a sensor for the redox state of the cell, it is mainly known to be activated by the above-mentioned DSB sensing MRN-complex (Bakkenist and Kastan, 2003; Falck et al., 2005; Kruger and Ralser, 2011; Perry and Tainer, 2011). ATR, on the other hand, is recruited to RPA-coated UV-induced lesions by the ATR INTERACTING PROTEIN (ATRIP) (Wright et al., 1998; Cortez et al., 2001; Ball and Cortez, 2005; Warmerdam et al., 2010). Once activated both kinases phosphorylate p53 and the effector kinases CHK1 and CHK2 regulating cell cycle and DNA repair (Brady and Attardi,

Curiously no plant homologues of p53 have been identified in any of the model organisms. This is probably linked to the absence of the core apoptotic machinery as we know it from animals. In contrast most of the DNA repair targets of p53, as well as ATM and ATR, are very well conserved in plants. Where loss of one of the checkpoint kinases in animals is lethal, the existence of viable *atr* and *atm* mutant plants in Arabidopsis make it an ideal model for their investigation. Both are involved in the response to ionizing radiation (IR) and necessary for the IR-induced transcription activation of many genes participating in DNA repair, cell cycle control, transcription, and replication (Culligan et al., 2006; Ricaud et

This raises the question if there is a factor that is functioning as a p53 analog mediating the DNA damage response between ATM/ATR and the downstream repair factors. An answer to that could be SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1). Though unrelated to p53 and unique to plants, this transcription factor, discovered in a screen for suppressor mutants of the -irradiation induced cell cycle arrest of Arabidopsis *uvh1* seeds, is necessary for the activation of genes downstream of both ATM and ATR in response to -irradiation (Preuss and Britt, 2003; Yoshiyama et al., 2009; Furukawa et al., 2010). SOG1, ATM, and ATR were also found to trigger plant programmed cell death (PCD) in root meristems after - or UV-B irradiation, a mechanism that was recently shown to be distinct from animal apoptosis (Fulcher and Sablowski, 2009; Furukawa et al., 2010). Hence, SOG1 is a good candidate to control repair processes in a p53-like fashion, at least by activating transcription of the plant homologues of factors like DDB2, MSH2

Current research indicates that plants and animals share roughly similar repair pathways. But for some repair proteins that have been described in animals no homologues have been found in plants, as yet. However, with ongoing research, it seems plausible that plant counterparts will be identified that can substitute for missing animal orthologs as it appears

Plants and animals share a surprisingly high degree of conservation among their abilities to repair damaged DNA (please also see Table 1 at the end of this passage for an overview of genes involved in DNA repair in the model plant *Arabidopsis thaliana*). While mammalian researchers have very valid and scientifically relevant reasons to use animal subjects, plants

al., 2007; Yoshiyama et al., 2009; Furukawa et al., 2010).

and XPC in response to UV and IR stresses.

**4. Plants as model organisms to study DNA repair** 

to be the case with p53 and SOG1.

Wiesmuller, 2006).

2010).

can and should be considered as excellent and viable alternatives to investigate the fundamentals of DNA repair processes. Tolerance towards mutations and abiotic stresses along with the relative ease of upkeep and propagation of the research organisms are two factors that we will briefly discuss in this final section of the review.

Due to their inability to elude many constantly damaging influences, plants need to utilize efficient ways to cope with these stresses. One strategy plants seem to have adopted to manage the higher demands on DNA repair is redundancy. For instance, genes of every pathway discussed here were found to be duplicated in Arabidopsis or rice (Singh et al., 2010). Additionally the existence of both 8-oxo-guanine glycosylases, OGG1, as well as MutM/Fpg in Arabidopsis demonstrate functional redundancy of independent, alternative repair pathways, which may have originated from the incorporation of chloroplast and mitochondrial genes into the nuclear genome (Boesch et al., 2009; Singh et al., 2010; Rowan et al., 2010).

Probably because of these gene duplications, functional redundancies, and more efficient or alternative pathways in comparison to animals, plants often have greater flexibilities in how they can respond to and potentially tolerate damaged DNA and mutations. For example a homozygous mutant in ATR kinase, which would be lethal in mammals, can in plants be investigated for the impact on DNA repair, control of apoptosis or gene expression profiles. In order to see the global effects of genotoxic stressors on a model organism, the subjects need to be exposed to different degrees of damaging agents. Here, plants are ideal models because of their sessile nature. They can be cultivated under very steady and reproducible conditions, while stress exposure is highly controlled. In addition, from an ethical point of view, plants can be taken to the edge of survival with very harsh treatments such as high levels of UV-light or toxin applications that for some may be not comfortable to perform on animals.

In comparison to animals, plants are low cost organisms that only require minimal monitoring along with water and occasionally fertilizer. Small plants like the moss *Physcomitrella patens* or *Arabidopsis thaliana* can be cultivated to great numbers within in a few square feet while by comparison animals require adequate space and regular food, water, and cleaning. While mutant lines are readily available for many animal and plant systems, shipment and propagation of plant resources can be quite straightforward. Seeds can be harvested for immediate propagation of the next generation or stored long-term, even at room temperature, before use months or even years later. Sending seed material to colleagues around the world is technically simple since no special transport accommodations need to be made. Generating transgenic *Arabidopsis* lines using *Agrobacterium* infection is a standard lab procedure, and allows for rapid complementation of mutant lines to verify protein functionality and observation of response and recovery. Also generation time of Arabidopsis plants is very short with just two months from seed to seed.

In addition to using plants as basic models to understand DNA repair processes, there are also practical reasons why this area of research urgently needs to be expanded. With the increase in food shortages for increasing populations, the recognition of environmental toxins and the growing evidence of impending and occurring climate changes across the world, it becomes critical to rapidly develop plants that can better cope with environmental stress. As such, stress tolerant crop plants generated either by genetic engineering or classical breeding will become increasingly important resources to guarantee stable food supplies to the human population in an expected changing environment.

Recognition and Repair Pathways of Damaged DNA in Higher Plants 217

**Arabidopsis Function Acc. No. Reference** 

Singh et al., 2010

Singh et al., 2010

Singh et al., 2010

Singh et al., 2010

Liu et al., 2003

Jenkins et al., 1995

Liu et al., 2003

Hellmann, 2010; Zhang et al., 2010

Zhang et al., 2010

2001; Dany and Tissier, 2001

2009

2009

AT1G73690 Singh et al., 2010

AT1G66750 Singh et al., 2010

AT1G18040 Singh et al., 2010

AT1G27840 Biedermann and

AT1G19750 Kunz et al., 2005;

AT2G18760 Shaked et al., 2006

AT1G21710 Garcia-Ortiz et al.,

AT1G52500 Ohtsubo et al., 1998

AT2G31450 Gutman and Niyogi,

*TFIIH2 AtGTF2H2* TFIIH subunit p44 At1g05050 Kunz et al., 2005;

*TFIIH3 AtTFB4* TFIIH subunit p34 AT1G18340 Kunz et al., 2005;

*TFIIH4 AtTFB2* TFIIH subunit p52 At4g17020 Kunz et al., 2005;

*TFIIH5 AtTFB5* TFIIH subunit AT1G12400 Kunz et al., 2005;

cyclin activating kinase-subcomplex of THIIH

substrate recognition for CUL4-dependent ubiquitination, TCR

polymerase, recruitment of repair machinery, TCR

glycosylase

formamidopyrimidine DNA glycosylase

apyrimidinic (AP) lyase/endonuclease

 *CHR24* AT5G63950 Shaked et al., 2006

*AtNTH2* AT1G05900 Gutman and Niyogi,

*CHR8* binding of stalled RNA

AT1G62886 Singh et al., 2010

*AtXPB1* helicase subunit of TFIIH AT5G41370 Costa et al., 2001

*UVH3/UVR1* 3'-endonuclease AT3G28030 Jenkins et al., 1995;

helicase subunit of TFIIH AT1G03190 Jenkins et al., 1995;

5'-endonuclease AT5G41150 Harlow et al., 1994;

 *AtXPB2* AT5G41360 Morgante et al., 2005

**Repair pathway**  **Representative Gene Model** 

*XPB/RAD25/ER CC3* 

*XPD/RAD3/ER CC2* 

*XPF/RAD1/ERC C4* 

*XPG/RAD2/ER CC5* 

*CSA/RAD28/ER CC8* 

*CSB/RAD26/ER CC6* 

**Gene in** 

*CDK7 CAK3AT/CD*

 *CAK4AT/CD*

 *CAK2AT/CD*

*KD1;1* 

*KD1;2* 

*KD1;3* 

*AtXPD/UVH 6* 

*AtRAD1/UV H1* 

*ATCSA-1/CSAat1A* 

*2/CSAat1B* 

**BER** *OGG1 AtOGG1* 8-oxoguanine DNA

*H* 

*NTH AtNTH1* DNA glycosylase and

 *ATCSA-*

*MutM AtFPG/MM*


6-4PPs, GGR

6-4PPs, GGR

6-4PPs, GGR

dependent ligases

6-4PPs, GGR, substrate recognition for CUL4 dependent ubiquitination

*TFIIH1 AtTFB1-1* TFIIH subunit p62 At1g55750 Kunz et al., 2005;

 *AtTFB1-2* At1g55680 Kunz et al., 2005;

 *AtTFB1-3* At3g61420 Kunz et al., 2005;

 *RAD23B* AT1G79650 Molinier et al., 2004b

 *RAD23C* AT3G02540 Molinier et al., 2004b;

 *RAD23D* AT5G38470 Molinier et al., 2004b;

 *RBX1B* AT3G42830 Lechner et al., 2002;

*CUL4 CUL4* Ubiquitylation of targets AT5G46210 Molinier et al., 2008;

*DDB1 AtDDB1a* Ubiquitylation of targets AT4G05420 Molinier et al., 2008;

 *AtDDB1b* AT4G21100 Bernhardt et al., 2006;

*RAD23A* recognition of CPDs and

*CEN2 AtCEN2* recognition of CPDs and

*DDB2 DDB2* recognition of CPDs and

*ROC1/RBX1 RBX1A* activation of CUL4-

**Arabidopsis Function Acc. No. Reference** 

*PHR1/UVR2* repair of CPDs AT1G12370 Ahmad et al., 1997;

*PHR2* repair of CPDs AT2G47590 Ahmad et al., 1998;

*UVR3* repair of 6-4PPs AT3G15620 Jiang et al., 1997;

Landry et al., 1997

Petersen et al., 1999

Nakajima et al., 1998

Liang et al., 2006

Kunz et al., 2005; Farmer et al., 2010

Kunz et al., 2005; Farmer et al., 2010

Kunz et al., 2005; Farmer et al., 2010

Kunz et al., 2005; Farmer et al., 2010

Liang et al., 2006

Gray et al., 2002

Gray et al., 2002

(Biedermann and Hellmann, 2010

(Biedermann and Hellmann, 2010

Bernhardt et al., 2010

Molinier et al., 2008

Singh et al., 2010

Singh et al., 2010

Singh et al., 2010

At5g58760 Koga et al., 2006;

AT4G37010 Molinier et al., 2004b;

AT5G20570 Lechner et al., 2002;

AT5G16630 Kunz et al., 2005;

AT1G16190 Molinier et al., 2005;

**Repair pathway** 

**Photoreactivation**  **Representative Gene Model** 

*HHR23A,B/RA D23* 

**Gene in** 

**NER** *XPC AtRAD4* recognition of CPDs and


Recognition and Repair Pathways of Damaged DNA in Higher Plants 219

pairing

processivity factor activity

poly(ADP)ribosylation

stabilization of singlestranded DNA intermediates

*RPA3* AT3G52630 Singh et al., 2010

Table 1. Overview of genes involved in DNA repair in the model plant *Arabidopsis thaliana*.

Aboussekhra, A., Biggerstaff, M., Shivji, M.K., Vilpo, J.A., Moncollin, V., Podust, V.N.,

Protic, M., Hubscher, U., Egly, J.M., and Wood, R.D. (1995). Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80,

*LIG1 AtLIG1* DNA ligation AT1G08130 Taylor et al., 1998

**Other** *ATM AtATM* checkpoint kinase AT3G48190 Garcia et al., 2000)

**Arabidopsis Function Acc. No. Reference** 

*AtPCNA2* AT2G29570 Strzalka et al., 2009

*AtPARP2* AT2G31320 Doucet-Chabeaud et

*AtPARP3* AT5G22470 Singh et al., 2010

*AtRPA1-1* AT4G19130 Kunz et al., 2005;

 *AtBRCA2B* AT5G01630 Siaud et al., 2004;

AT4G00020 Siaud et al., 2004;

AT1G07370 Strzalka et al., 2009

AT1G49250 Singh et al., 2010

AT4G02390 Lepiniec et al., 1995

AT2G06510 Ishibashi et al., 2005;

AT5G08020 Kunz et al., 2005;

AT5G45400 Kunz et al., 2005;

AT5G61000 Kunz et al., 2005;

AT2G24490 Kunz et al., 2005

AT3G02920 Kunz et al., 2005

AT4G18590 Singh et al., 2010

checkpoint kinase AT5G40820 Culligan et al., 2004

Dray et al., 2006

Dray et al., 2006

al., 2001

Chang et al., 2009

Singh et al., 2010

Singh et al., 2010

Singh et al., 2010

Singh et al., 2010

**Repair pathway**  **Representative Gene Model** 

**Gene in** 

*D3* 

*ATR/RAD3 AtATR/AtRA*

*RPA1 AtRPA1A/At*

*RPA2 AtRPA2-*

**5. References** 

859-868.

*RPA1- 3/RPA70A* 

*AtRPA1- 5/RPA70B* 

*AtRPA1- 2/RPA70C* 

*AtRPA1- 4/RPA70D* 

*1/ATRPA32 A* 

*AtRPA2- 2/ATRPA32B*

*BRCA2 AtBRCA2A* supports homology

*PCNA AtPCNA1* DNA polymerase

*PARP AtPARP1* damage recognition,


(AP) endonuclease

strand break ends

strand break ends

complex, damage recognition, generation of single-stranded DNA

complex, damage recognition, generation of single-stranded DNA

complex, damage recognition, generation of single-stranded DNA

DNA binding, mediation of inter-strand-pairing

DNA binding, mediation of inter-strand-pairing

pairing

 *AtRECA2* AT2G19490 Shedge et al., 2007  *AtRECA3* AT3G10140 Khazi et al., 2003;

AT3G32920 Shedge et al., 2007  *DRT100* AT3G12610 Pang et al., 1992;

 *RAD51B* AT2G28560 Bleuyard et al., 2005;

 *RAD51C* AT2G45280 Bleuyard et al., 2005

*XRCC4 XRCC4* co-factor of DNA ligase 4 AT3G23100 West et al., 2000 *LIG4 AtLIG4* DNA ligation AT5G57160 West et al., 2000

*XRCC1 AtXRCC1* co-factor of DNA ligase 3 AT1G80420 Petrucco et al., 2002;

*FEN FEN1* flap endonuclease AT5G26680 Singh et al., 2010

**Arabidopsis Function Acc. No. Reference** 

*APE2* AT4G36050 Singh et al., 2010

AT2G41460 Babiychuk et al.,

AT1G16970 Riha et al., 2002

AT1G48050 Riha et al., 2002

AT5G54260 Hartung and Puchta,

AT2G31970 Gallego and White,

AT3G02680 Bleuyard et al., 2006;

AT1G79050 Cerutti et al., 1992;

1994; Gutman and Niyogi, 2009

Singh et al., 2010

1999; Daoudal-Cotterell et al., 2002

2001

Waterworth et al., 2007

Cao Cao et al., 1997; Shedge et al., 2007; Rowan et al., 2010

Shedge et al., 2007

Pang et al., 1993

Osakabe et al., 2005

Osakabe et al., 2006

Montane, 2003

AT5G20850 Doutriaux et al., 1998

AT1G07745 Bleuyard et al., 2005;

AT4G21070 Lafarge and

**Repair pathway**  **Representative Gene Model** 

**Gene in** 

**NHEJ/HR** *KU70/XRCC6 AtKU70* binding of DNA double

*KU80/XRCC5 AtKU80* binding of DNA double

*MRE11 AtMRE11* subunit of the MRN

*RAD50 AtRAD50* subunit of the MRN

*NBS1 AtNBS1* subunit of the MRN

*RecA* 

*AD51A* 

*N1* 

*BRCA1 AtBRCA1* supports homology

*RECA (E. Coli) AtRECA1/cp*

*RAD51 AtRAD51/R*

 *RAD51D/SS*

*APE ARP/APE1* apurinic/apyrimidinic


Table 1. Overview of genes involved in DNA repair in the model plant *Arabidopsis thaliana*.
