**5. Conclusions**

36 DNA Repair

plant tRNA splicing ligase (Abelson *et al*, 1998). What makes this repair system unique is its ability to render the restored phosphodiester linkage immune to re-cleavage by virtue of the 2'-O methylase activity of Hen1 (Chan *et al*, 2009b). PnkP comprises an N-terminal kinase domain, a central metallophosphoesterase domain and a C-terminal ligase domain. Thus, it comprises functions similar to those of the yeast tRNA splicing ligase but differs in domain order and different origin of the phosphoesterase domain (Apostol *et al*, 1991;Martins & Shuman, 2005). Interestingly, by itself the bacterial PnkP heals 2', 3'-cyclic P and 5'-OH termini pairs and undergoes the first step in the RNA ligase reaction, its auto-adenylation, but does not proceed to activate the 5'-P end and generate the phosphodiester linkage (Martins & Shuman, 2005). This deficiency is corrected by expressing PnkP with the 2'-O methylase Hen1. Within the resultant PnkP/Hen1 complex PnkP heals and seals the cleavage termini while Hen1 2'-O methylates the dephosphorylated 3'-end prior to the ligation step. This modification renders the restored ligation junction immune to recleavage (Chan *et al*, 2009b). The bacterial Hen1 is so named because it resembles in sequence and structure the methylase domain of eukaryal miRNA methyltransferase Hen1 (Chan *et al*, 2009a). The eukaryal Hen1 protects the 3'-terminal ribose of miRNA from

exonucleolytic degradation or utilization as replication primer (Chen, 2005).

2002) have not been identified yet in bacteria likely to accommodate PnkP/Hen1.

**4.3 An essential eukaryal DNA repair protein is related to T4 Pnk** 

As with bacterial RtcAB, the biological role of the PnkP/Hen1 is not known. Noteworthy in this regard is that PnkP/Hen1 is most abundant among *Actinobacteria.* In contrast, RtcAB is more prevalent among *Proteobacteria* and has not been detected yet in *Actinobacteria*. This coincidence raises the possibility that the two systems provide similar benefits to their respective hosts. In theory, PnkP/Hen1 complexes could defend their host cells from secreted ribotoxins more efficiently than RtcAB due to the ability to prevent re-cleavage of the susceptible RNA. It is noteworthy though that colicin-like ribotoxins that target rRNA (Bowman *et al*, 1971;Senior & Holland, 1971) or tRNA anticodon loops (Masaki & Ogawa,

If PnkP/Hen1 were to counteract an ACNase that cleaves its substrate 3' to the wobble base like colicin E5 (Ogawa *et al*, 1999), then the repaired tRNA would contain a 2'-O methylated wobble nucleotide. Such a protective modification need not impair the tRNA's function since it exists in some natural bacterial tRNAs (Juhling *et al*, 2009). However, it cannot be excluded that PnkP/Hen1 plays additional or other roles and may be exploited differently in different bacterial hosts. One example of such a different role is hinted at by the juxtaposition of the PnkP/Hen1 and CRISPR-Cas loci of *Microscilla marina*. The CRISPR-Cas system confers adaptive immunity against foreign nucleic acids. During its antiviral interference activity specific RNA portions of the CRISPR transcript are used to target a Cas protein to cleave the invasive nucleic acid (Deveau *et al*, 2010). Hence, it may be asked if *M. marina* PnkP/Hen1 catalyze some RNA processing and/or modification steps during CRISPR RNA maturation. Finally, in a reversal of roles, one could envisage PnkP/Hen1 encoding phage able to prevent re-cleavage of a tRNA by the ACNase they counteract.

There are a number of examples of DNA repair devices that could have originated from RNA-specific progenitors, some of them already alluded to above. Here it will suffice to describe just one of them, related to the phage T4-encoded end healing protein Pnk. This conserved eukaryal protein termed interchangeably PNKP and Pnk1 contains 5'-kinase and 3'-phosphatase domains resembling those of T4 Pnk but arranged in the reverse order, the phosphoesterase domain preceding the kinase domain. The mammalian PNKP is also In this chapter we addressed the possible biological role of the conserved bacterial anticodon nuclease RloC that combines two seemingly conflicting properties. One, predicted by resemblance of its regulatory region to the universal DNA-damage-checkpoint/DNA repair protein Rad50/SbcC is monitoring DNA insults. The second, predicted by its tRNase activity is disabling the translation apparatus. The co-existence of such functions in the same molecule and the regulation of one by the other suggests that RloC is designed to block translation in response to DNA damage. Such a response is suicidal since it prevents recovery from DNA damage. Hence, it must be executed only under special circumstances where cell death is advantageous. One possibility considered here is that RloC benefits its host cell by acting as an antiviral contingency during recovery from DNA damage. Under these conditions bacterial cells may shut off their primary antiviral defense, i.e., their DNA restriction activity. RloC's suicidal activity would not rescue the infected cell but would prevent the spread of the infection to other vulnerable members of the population recovering from DNA damage.

Another unique property, which could make RloC particularly suited to thwart phage infection, is the ability of this ACNase to excise its substrate's wobble nucleotide. In this regard RloC differs from its distant homologue the ACNase PrrC, which only incises its tRNA substrate and is counteracted by phage tRNA repair enzymes. Therefore, it seems conceivable that the harsher lesion inflicted by RloC will encumber such phage reversal. The possibility that RloC is a more efficient antiviral device than PrrC is also hinted at by its ~3-fold more frequent occurrence among bacteria.

While these notions are supported by some demonstrated properties of RloC, testing them and identifying RloC's true call requires studying this protein under physiological conditions; ideally, using a natural host encoding it and cognate phages endowed with tRNA repair enzymes.

The RNA repair pathway instigated by PrrC and possibly avoided by RloC brings to the fore the rather overlooked issue of RNA-damage-repair. Such repair would seem necessary only under circumstances such as the absence of a DNA template to transcribe from. Nonetheless, recent discoveries of various cellular RNA repair devices distributed in the three domains of life suggest that RNA damage repair is more prevalent, exercised perhaps also during

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**3** 

Chuck C.-K. Chao

 *Republic of China* 

**The Role of DDB2 in Regulating Cell** 

*Graduate Institute of Biomedical Sciences, Chang Gung University, Taiwan* 

Nucleotide excision repair (NER) represents a central cellular process for the removal of structurally and chemically diverse DNA lesions [Friedberg et al., 2006]. Mutations in genes involved in NER are associated with rare autosomal recessive syndromes such as xeroderma pigmentosum (XP), a condition characterized by sensitivity to UV light, neurological abnormalities, and a propensity to develop skin cancer (Cleaver, 2005). The observation that cells from XP subgroup E (XP-E cells XP2RO and XP3RO) are defective in recognizing damaged DNA and performing NER highlighted the physiological importance of the protein termed DNA damage-binding protein, or DDB [Chu & Chang, 1988]. The DDB protein, sometimes also referred to as UV-DDB due to its high affinity and specificity for UV-damaged DNA, contains two principal subunits, DDB1 and DDB2 [Grossman, 1976; Keeney et al., 1993; Takao et al., 1993]. The DDB protein complex also binds to non-UVdamaged DNA, like cisplatin-modified DNA, although with much lower affinity. Although the history of DDB spans more than two decades, the complete understanding of its physiological functions remains to be clarified. The activity of DDB has been repeatedly described in crude mammalian cell extracts by electrophoretic mobility shift assays or filterbinding assays performed by different laboratories since the first report of its discovery [Feldberg & Grossman, 1976]. Notably, micro-injections of DDB complexes into the nucleus of XP-E cells restored NER activity [Keeney et al., 1994], supporting the notion that DDB participates in chromatin NER. The *DDB1* gene from simian cells was the first *DDB* gene to be identified [Takao et al., 1993]. The human *DDB1* and *DDB2* genes were subsequently sequenced [Dualan et al., 1995; Lee et al., 1995]. Soon after, DNA sequencing from Linn's laboratory revealed that *DDB2* is mutated in XP-E cells which lack DDB activity [Nichols et al., 1996; Tang & Chu, 2002]. The predicted DDB2 protein sequence was shown to contain several functional domains, including WD40 repeats, post-translation modification sites (e.g. acetylation, phosphorylation, and ubiquitination), DDB1- and DNA-binding sites, as well as a DWD box. Notably, in a majority of XP-E cell lines, DDB2 was found to be altered at domains other than the one required for binding DNA. Thus, DDB appears to be regulated at several levels in UV-irradiated cells, including by transcriptional activation of DDB2 mRNA, post-translational modification, translocation to the nucleus, complex formation,

**1. Introduction** 

**Survival and Apoptosis Following** 

**DNA Damage - A Mini-Review** 

*Department of Biochemistry and Molecular Biology* 

