Técnicas de Procesamiento, Análisis de Datos y Presentación de Datos

Studies of E. coli Rep protein led to the first proposal of the ‘inchworm’ mechanism of strand unwinding (Yarranton and Gefter, 1979). This model, however, has been refined since Rep was identified as a homodimer upon DNA binding (Wong et al., 1992; Lohman and Bjornson, 1996).

The ‘inchworm’ model requires coordinated alternate binding of the nucleic acid at two different sites within the functional unit of the helicase. One site is required to bind ssDNA (tail site) and the other to bind both ssDNA and duplex DNA (leading site) (Lohman and Bjornson, 1996). This can be accomplished by a monomer containing two binding sites, or a dimer with a single binding site per subunit. Translocation along the ssDNA is coupled to ATP binding, whereas the unwinding reaction is driven by ATP hydrolysis (Tuteja and Tuteja, 2004).

Structural characterisation of Bacillus subtilis PcrA has led to a more detailed mechanistic view of DNA unwinding by the ‘inchworm’ model (Mackintosh and Raney, 2006). The helicase reaction can be divided into two processes, the translocation of PcrA along ssDNA and duplex destabilisation. It is the coupling of these that constitutes helicase activity (Velankar et al., 1999).

Initially ssDNA is bound to both domains 1A and 2A. Upon ATP binding the conformation of PcrA changes and it adopts a ‘closed’ arrangement. At this stage domain 2A is bound to the ssDNA and 1A has loosened its grip to slide along the strand. Hydrolysis of the ATP causes domain 1A to grip the DNA tightly, now 2A releases the strand and translocation is affective across it. The protein is said to be in an open conformation (Velankar et al., 1999). This process by which tight DNA binding alternates between the two domains is described in cartoon form in figure 1.9A.

A

B

When domain 1A has a tight grip on the DNA, bases are in each of the acceptor pockets of 1A (figure 1.9B). When PcrA adopts the ‘closed’ conformation, domain 1A releases its grip on the DNA by displacement of bases in its binding pockets; the result is translation across domain 1A. Flipping of the adjacent bases into the binding pockets of domain 1A promotes tight binding of domain 1A once again to the ssDNA strand and the DNA is pulled over domain 2A (Velankar et al., 1999). Step sizes of only a few to as little as one base pair are characteristic of the ‘inchworm’ unwinding mechanism (Velankar et al., 1999).

SF2 helicases are mechanistically distinct from other helicases. In contrast to SF1 helicase that rely on the bases as the primary recognition determinant, SF2 helicases can track along the continuous ribose-phosphate backbone of the loading strand without

Figure 1.9 Proposed models for the translocation of PcrA along ssDNA

A) The affinity of each of the domains of PcrA, 1A and 2A, alternates during translocation and is dependent upon ATP binding or hydrolysis. ATP binding induces a closed cleft conformation, which allows 2A to bind tightly to the DNA. Upon ATP hydrolysis, the cleft opens. 2A loosens its grip on the DNA and PcrA springs back to its original conformation. 1A retains a tight grip on the DNA at this point pulling the DNA across domain 2A.

B) A cartoon representation of the base flipping events that occur during translocation of PcrA along ssDNA. Domain 1A is tightly bound to the DNA when there is a base in binding pocket B. The side chain of F64 moves into this pocket displacing the base to pocket A, which in turn displaces the adjacent base from pocket A to the outside of the protein. ATP hydrolysis opens the cleft, now F64 is released and the base in pocket C can move to pocket B. Cleft opening now forces the base from the stacked pair in pocket D to flip to pocket C, the second of this pair moves along and the next base can join it. Both figures were adapted from Velankar, et al. (1999).

interacting with the bases. RNA helicase NPH-II is an example of a helicase that peels the complementary strand off during translocation (Kawaoka et al., 2004).

1.5.1.1‘Cooperative Inchworm’ model

Some proteins, despite being monomeric, exhibit enhanced unwinding when multiple monomers are allowed to function cooperatively. The basis of the ‘cooperative inchworm’ model describes independently translocating monomers that cooperate upon encountering a ‘challenge’, for example, duplex DNA or a protein block. Multiple monomers increase the chances of overcoming such an obstacle and the more difficult the challenge, the more important this enzyme cooperativity becomes (Mackintosh and Raney, 2006). The bacteriophage T4 Dda helicase is an example of a helicase that exhibits greater helicase activity when the single stranded region of the DNA is long enough to accommodate at least two monomers of Dda. The increased activity, however, is most likely not the result of protein-protein interactions but instead simply due to the presence of other monomers, translocating in the same direction, that can prevent the first monomer slipping backwards upon encountering a block (Byrd and Raney, 2004, 2006). 1.5.1.2‘Quantum Inchworm’ model

The ‘quantum inchworm’ model describes an unwinding mechanism by which the leading domain (L) of the helicase binds one strand of the duplex DNA ahead of the trailing domain (T), anchoring the protein to the duplex. The trailing domain advances, unwinding the DNA in small steps. As the trailing helicase domain approaches the anchored leading domain it transmits a signal, which leads to dissociation and rebinding of the leading domain to the duplex DNA ahead to restart the process (Bianco and Kowalczykowski, 2000). E. coli RecBC helicase is an example of an enzyme that catalyses DNA unwinding according to this model (figure 1.10)