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Helicases have a major involvement in DNA metabolism, including transcription, recombination and repair, and have been linked to a number of human genetic disorders. In humans, defects in the RecQ family helicases WRN, BLM and RecQ4 are characterised by genomic instability and an increased incidence of cancer. They are associated with the rare autosomal disorders Werner’s Syndrome (WS), Bloom’s Syndrome (BS) and Rothmund Thomson Syndrome (RTS), respectively (Bennett and

Keck, 2004; Opresko et al., 2004). Helicases function to catalyse nucleic acid duplex separation and this action is dependent upon the hydrolysis of a nucleoside 5’- triphosphate (NTP) (Hall and Matson, 1999). A helicase will usually bind to single stranded DNA or to a ssDNA/dsDNA junction and will translocate unidirectionally along the strand it has loaded onto. On the basis of the direction of translocation they can be divided into 2 types, 5’-3’ or 3’-5’ helicases. The protein sequences contain conserved motifs, found clustered in the ‘core’ region of the three-dimensional protein, that act as the engine to power this unwinding activity (Hall and Matson, 1999).

The presence of these motifs, however, is not sufficient to identify helicases since only a minority of these proteins actually possess true helicase activity (Singleton and Wigley, 2002). This may be because these proteins require oligomerisation, protein interactions or activation such as phosphorylation, for example. It is also true that these motifs are characteristic of translocases as the molecular motors themselves may not be enough to achieve strand separation and this may be the function of additional domains (Singleton and Wigley, 2002). Proteins involved in chromatin remodelling have been shown to contain the helicase motifs in their sequence as they possess the ability to disrupt the interactions between DNA and histones rather than unwind duplex DNA. It is, therefore, widely accepted that these motifs are typical of NTPases rather than helicases specifically.

1.4.1 The organisation of helicases into families

The helicases were originally classified into three superfamilies SF1, SF2, SF3 and two smaller families F4 and F5, based on the similarity and organisation of conserved ‘signature motifs’ and primary structure analysis (Gorbalenya and Koonin, 1993; Hall and Matson, 1999; Tuteja and Tuteja, 2004). More recently it has become apparent that not all of the putative helicases catalyse strand separation. Instead, they couple NTP hydrolysis to translocation along nucleic acids. In addition, it has been suggested that the AAA+ ATPase proteins are defined as Superfamily 6 (SF6) since many nucleic acid motors are members of this family (Singleton et al., 2007). Consequently, the helicases are defined by the six superfamilies shown in figure 1.7. The N- and C-terminal regions show much sequence and length variability, for this reason these regions are thought to

be responsible for the individual functions exhibited by the helicases (Tuteja and Tuteja, 2004).

Superfamilies 1 and 2 are the largest of the families and are closely related (figure 1.7). Helicases from both families contain the seven typical short amino acid sequence fingerprints, which are very similar in sequence, arrangement and secondary structure (Hall and Matson, 1999). These conserved helicase motifs are usually clustered in a core region of 200-700 residues in the amino acid sequence (Hall and Matson, 1999; Tuteja and Tuteja, 2004). The tandem RecA like folds found either in the same polypeptide or between subunits are formed from core domains. The Walker A and B boxes, involved in nucleotide binding and hydrolysis, and an arginine finger that plays a role in energy coupling are universal features of these core domains (Singleton et al., 2007). Many ATPases share this common core RecA like fold, which contains the ATP binding site. This fold was initially identified in E. coli RecA protein and is, therefore, referred to as the RecA-like fold or RecA motor domain (Ye et al., 2004; Bell, 2005). Two RecA-like domains are found in the SF1 and SF2 helicases but this number varies among different ATPases (Story et al., 1992; Ye et al., 2004; Singleton et al., 2007). SF3-6 members

Figure 1.7. Classification of NTPases into six Superfamilies

Nucleic acid motors are classified into six Superfamilies based on specific core motifs. These core domains and the signature motifs for each family are shown, however, the precise position is based on the example shown and is representative of the whole family. The positions and functions of the accessory domains are shown but these are specific for this particular example, their location, function and even presence may vary between members of the same family. Universal structural elements common to all helicases are shown in yellow (Singleton et al., 2007).

form hexameric or double hexameric rings and, therefore, possess six or twelve RecA- like folds (Singleton et al., 2007).

The proximity of all of the motifs to each other and the NTP implicates them all in nucleotide binding and hydrolysis and possibly the coupling of energy from hydrolysis to DNA unwinding, despite not all motifs making physical contact with the bound nucleotide (Hall and Matson, 1999). Structural data for the two SF1 helicases, B. stearothermophilus PcrA and E. coli Rep, has shown that the seven motifs occupy positions in domains 1A and 2A of the four-domain proteins and that these two domains are located at the base of the enzymes forming a nucleotide binding pocket (Hall and Matson, 1999). Motifs I, Ia, II, V and VI of the SF2 helicase NS3 appear to occupy the same relative positions compared to the SF1 helicases and they fulfil similar functions in both superfamilies (Hall and Matson, 1999). Only motifs III and IV show major differences between the NS3 and the two SF1 proteins Rep and PcrA. The motifs are, therefore, well conserved across the two superfamilies to act together as the engine to power the enzyme. Both SF1 and SF2 contain ssDNA 3’-5’ and 5’-3’ motors, however, no dsDNA motors are found in SF1. The specific function of each helicase is achieved by a degree of variation between proteins (Hall and Matson, 1999). The XPB proteins and Hel308 from S. solfataricus are members of SF2 and are discussed in detail in chapters 3 and 5, respectively.

The helicases categorised into SF3 are limited to those found in DNA and RNA viruses, this group is much smaller than SF1 and 2. They form hexameric helicases that translocate along DNA (3’-5’) threading the ssDNA through the central channel of the ring (Singleton et al., 2007). The members of SF3 contain fewer conserved motifs in their peptide sequence; the Walker A and B boxes, the B’ box and the sensor 1 motif. Based on data obtained for AAV type 2 (AAV2) Rep40, it was proposed that the protein must oligomerise in order to form a catalytic nucleotide-binding site (James et al., 2004). Therefore, ATP binding acts to regulate the oligomeric state of this helicase. This may be a characteristic of SF3 helicases (James et al., 2004). The B’ motif, found only in SF3 helicases, is important for the helicase reaction since it encompasses residues involved in direct interactions with the nucleotide at the oligomeric interface and has been proposed to bind ssDNA necessary for helicase activity (Koonin, 1993; Yoon-Robarts et al., 2004).

Motif B’ couples DNA binding and ATP hydrolysis and possesses residues with a direct involvement in DNA interactions (James et al., 2004).

The helicases of the fourth family are related to bacterial DnaB. They are associated with a primase, both functionally and physically, either as two distinct proteins that interact or as two domains of the same protein (Gorbalenya and Koonin, 1993). They contain only five conserved motifs (H1, H1a, H2, H3 and H4), including the Walker A (H1) and B (H2) boxes, and unwind DNA by threading the single stranded portion through the centre of a hexameric ring structure (5’-3’) (Caruthers and McKay, 2002; Donmez and Patel, 2006). The most extensively studied SF4 helicase is bacteriophage T7 gene 4 protein (T7gp4) the helicase mechanism of which is discussed in section 1.5.3.

There is also a fifth group that contains helicases (5’-3’) such as Rho, the bacterial transcription terminator factor (Singleton and Wigley, 2002). Rho is a DNA-RNA helicase with significant sequence similarity to proton translocating ATPase, rather than other helicases (Gorbalenya and Koonin, 1993). SF6 includes proteins such as the MCMs that contain the AAA+ fold but do not fall into SF3 (Singleton et al., 2007). 1.4.2 The helicase ‘signature’ motifs of SF1 and SF2 helicases

There is strict conservation of the Walker A and Walker B box sequences across the helicases families, motif VI is the third most conserved, whereas motifs Ia, III and V show much more sequence variation among different families (Gorbalenya et al., 1989; Tuteja and Tuteja, 2004).

The Walker A box (motif I) is identified by the consensus sequences AxxGxGKT (SF1) and GxxxxGKT/S (SF2); the final three residues are the minimal requirement (Gorbalenya et al., 1989). The side chain of the invariant lysine contacts the β-phosphate of the NTP upon binding to stabilise the transition state during the hydrolysis reaction (Gorbalenya et al., 1989; Hall and Matson, 1999; Caruthers and McKay, 2002; Tuteja and Tuteja, 2004). The Walker B box (Motif II) generally takes the form of DExx across the two major superfamilies. Proteins in SF2 with this motif are also referred to as the DEAD-box proteins, as their signature motif consists of the consensus Asp-Glu-Ala-Asp (Gorbalenya et al., 1989; Pause and Sonenberg, 1992). The Walker B box is thought to activate the attacking water molecule and, more specifically, the carboxyl portion of the

aspartic acid coordinates the Mg2+

ion in order to do this. The glutamic acid is important for ATP hydrolysis (Hall and Matson, 1999; Caruthers and McKay, 2002).

The sequence of motif VI exhibits covariation with the Walker B box; proteins with the sequence DEAD in motif II have the signature ‘HxxGRxxR’ in motif VI and those with ‘DExH’ consensus have ‘QxxGRxxR’ (Gorbalenya et al., 1989). Motif VI is responsible for mediating nucleotide binding induced conformational changes.