ESTILO DE VIDA SALUDABLE “DESCANSO Y SUEÑO”

The crystal structure of E. Coli thymine glycol DNA glycosylase (EndoIII) indicated a

novel DNA binding motif, which is called the Helix-hairpin-Helix (HhH) motif (Thayer, Ahern et al. 1995). The HhH motif has a length of ~20 amino acids (Doherty,

Serpell et al. 1996), consist two α-helices linked by a short hairpin loop which having

the coincidental sequences (Phe/Leu)-Pro-Gly-(Ile/Val)-Gly. This motif presents in many DNA-binding proteins such as DNA polymerases and DNA glycosylases (Nash, Bruner et al. 1996). The motif binds DNA in a sequence independent manner (Doherty, Serpell et al. 1996) and it is considered as the key elements that involved in interactions with DNA (Sawaya, Prasad et al. 1997; Rafferty, Ingleston et al. 1998).

At least six different DNA glycosylases have been identified currently are the members of the HhH DNA glycosylase family, and each of them has multiple homologues (Doherty, Serpell et al. 1996; Dodson and Lloyd 2002; Dizdaroglu 2005). Although the sequence identity is low (~10 %) among the HhH DNA glycosylase family members (Drohat, Kwon et al. 2002), they all exhibit the signature HhH motif for DNA-binding. The hairpin loop of the HhH motif exhibits the signature sequence pattern (Phe/Leu)-Pro-Gly-(Ile/Val)-Gly, indicates that these DNA glycosylases may share a common mechanism for DNA-binding (FIGURE 2.4)

FIGURE 2.4: Sequence alignments of HhH family members. Alignment using BioEdit (Hall 1999) and DALI (Holm and Sander 1993) server. Identical residues in the sequences aligned are highlight in black, residues shaded grey indicate regions of sequence homology. The red box shows the conserved residues from the proposed DNA binding motif (HhH motif). The TAG, hOGG, AlkA, EndoIII and MutY each corresponds to 3-MeA DNA glycosylase I, 3-MeA DNA glycosylase II, thymine glycol DNA glycosylase and adenine-specific mismatch-DNA glycosylases.

Comparative studies on these HhH DNA glycosylases have provided valuable information about enzymatic mechanism and helped identify the critical amino acids

that are essential for DNA and ligand binding such as 3-MeA DNA glycosylase II (AlkA) (Labahn, Scharer et al. 1996; Hollis, Ichikawa et al. 2000). The crystal

structure of E. coli AlkA in complex with DNA revealed that it used an activated

water nucleophile to cleave to adenine mispaired with guanine (Guan, Manuel et al. 1998; Hollis, Ichikawa et al. 2000). This proposed mechanism is similar to adenine-specific mismatch-DNA glycosylases (MutY) (Fromme, Banerjee et al. 2004). Crystal structure of the human 8-oxoguanine DNA glycosylase (Bruner, Norman et al. 2000) also shows a similar DNA-binding mechanism to EndoIII, they both use a catalytic residues Lys for the nucleophile attack (Mol, Kuo et al. 1995; Thayer, Ahern et al. 1995).

FIGURE 2.5: The HhH motif of DNA glycosylases. Schematic representation of the HhH subfamily DNA glycosylases structures. Helices are shown as grey cylinders and the HhH structure motif are shown in blue.

Enzyme Source/Gene References

Alkylbase-DNA glycosylases E. Coli (TAG) E. Coli (AlkA) H. Pylori (MagIII)

(Drohat, Kwon et al. 2002) (Labahn, Scharer et al. 1996) (Eichman, O'Rourke et al. 2003) Adenine-specific mismatch-DNA

glycosylases

E. Coli (MutY) (Guan, Manuel et al. 1998) DNA glycosylases removing

oxidized pyrimidines

E. Coli (EndoIII) (Kuo, McRee et al. 1992) DNA glycosylases removing

oxidized pyrines

S. Cerevisiae (OGG) (Bruner, Norman et al. 2000)

2.2.4

3-Methyladenine DNA glycosylase I (TAG) from S. aureus

Alkylated bases such as 3-MeA, 3-MeG and 7-MeG are cytoxic and mutagenic DNA lesions (Klungland, Fairbairn et al. 1992; Klungland, Bjoras et al. 1994), failure to repair leads to the stalling of regular duplication of DNA (Boiteux, Huisman et al. 1984), leading to more serious problems. Lesion repair begins by enzymatic hydrolysis of the glycosidic bond in DNA (the critical initial step of the BER pathway). The glycosidic bond of alkylated purine bases is usually hydrolyzed by two alkyl purine specific DNA glycosylases, 3-Methyladenine DNA glycosylase I (TAG) (Lindahl 1993) and II (AlkA) (Wyatt, Allan et al. 1999) (FIGURE 2.6).

FIGURE 2.6: Schematic representation of BER intermediates results DNA strand breaks. Picture modified from (Memisoglu and Samson 2000)

smallest member of the HhH family of DNA glycosylases (~186 residues, Mw: ~21 kDa) and has been shown that have a fairly narrow ligand specificity for the alkylated base, 3-MeA (Drohat, Kwon et al. 2002; Cao, Kwon et al. 2003). TAG shows very low overall protein sequences similarity compared with other HhH family DNA

glycosylases (~8%). In S. aureus TAG, the highly conserved Pro-Gly-(Val/Ile)-Gly

sequence pattern is not observed, replaced by Lys/Val-Phe-Val/Ile-Gly. Despite the

low identity of overall sequences, S. aureus TAG still shows the conserved sequence

motif at the HhH domain (FIGURE 2.7).

FIGURE 2.7: Sequence alignment of the the HhH motif in TAGs. The Highly conserved hairpin loop in HhH motif is highlighted by the red box.

The crystal structure of S. aureus TAG in complex with its product would provide

valuable information to understand the DNA binding and base excision mechanism. There was no crystal structure of TAG bound to its product when we carried out this

study, only a solution structure (NMR) of the E. coli TAG and an apo-form of TAG

from Salmonella typhi (Cao, Kwon et al. 2003) (FIGURE 2.8). Maintaining of DNA

is essential for the survival and evolution of S. aureus, making TAG a possible drug

target. The protein is up-regulated in MRSA476, but down-regulated in MSSA252. Such proteomic differences implying that this protein may be important for the pathogenic strain. Structural study on this protein binding to its product and DNA will help us better understand the catalytic mechanism, and also contribute to inhibitor

design.

FIGURE 2.8: Cartoon representation of the NMR structure of [A] E. coli TAG (PDB: 1P7M) and crystal structure of [B] Salmonella. Typhi TAG (PDB: 2OFK). Zn2+ ion is shown in grey sphere.