CAPÍTULO IV. RESULTADOS
4.1. Comprobación de la hipótesis general determinación de los indicadores
Hel308 has been shown here to exhibit 3’-5’ and 5’-3’ translocase and helicase activity. It is possible, therefore, that in vivo this could be a property that is governed by the strand or the orientation by which Hel308 is loaded onto the DNA (figure 5.17). Positioning of Hel308 on the lagging strand template would be appropriate for unwinding the nascent lagging strand (3’-5) or unwinding the parental duplex (5’-3’). Alternatively, if Hel308 loaded onto the leading strand template the nascent leading strand (5’-3’) would be displaced or, once again, the parental duplex (3’-5’).
Since the truncation mutant K646STOP was able to unwind all of the substrates to a certain extent, domain 5 could have a vital role in the correct positioning and orientation of Hel308 to ensure the correct strand is unwound. There may, also, be a requirement for interacting proteins to ensure correct loading of Hel308 and to alleviate the block imposed by domain 5. Alternatively, there may be an obligation for Hel308 to
Figure 5.17. A cartoon of the potential orientations of Hel308 on the DNA
Hel308 can catalyse 3’-5’ and 5’-3’ translocation and duplex unwinding. Therefore, the strand and orientation by which Hel308 is loaded onto the DNA may be vital for the appropriate function. Hel308 could load onto the lagging strand (3’-5’) or leading strand (5’-3’) template and displace the nascent lagging strand or leading strands, respectively. Alternatively, Hel308 could load onto the strands in the opposite orientations and, in both cases, destabilise the parental duplex.
partially unwind the parental duplex before removing the lagging strand, although this requires further investigation in order to fully understand the nature of this pattern of unwinding.
Hel308 exhibited the most efficient helicase activity, in vitro, when stimulated by a stalled replication fork structure with a corresponding lagging strand or a 3’ overhang structure. The schematic in figure 5.18 is a model proposed based on the results presented in this chapter and those from previous biochemical analysis (Guy and Bolt, 2005; Fujikane et al., 2006).
A lesion on the leading strand template causes the replication fork to stall, resulting in a gap since the leading strand cannot be primed downstream of the start site (Gregg et al., 2002). Similar to bacterial RecBCD and PriA at a replicative helicase block, it is thought that Hel308 is targeted to the stalled fork. The DNA displacement model describes the process by which Hel308 displaces the nascent lagging strand clearing the template for ssDNA binding proteins, for example RadA.
The E. coli RecFOR complex is known to remove ssDNA binding protein SSB from the nascent strand template and promote binding of RecA, which forms a nucleofilament on the DNA capable of initiating strand exchange (Umezu et al., 1993; Morimatsu and Kowalczykowski, 2003). As described here in the protein displacement mode (figure 5.18) an additional function of Hel308 may be to remove proteins from the ssDNA, since Hel308 was able to displace streptavidin from biotinylated ssDNA. Analogous to the proposed RecFOR pathway where SSB is displaced to allow RecA to bind, Hel308 could dislodge archaeal SSB (RPA in eukaryotes) from the DNA clearing the strand for binding of RadA (Rad51 in eukaryotes). This could form a nucleofilament, an active strand exchange species. The collinear relationship of the duplex substrate and the ssDNA product lends itself to many functions, including stripping proteins from DNA, since this mechanism is very versatile (Buttner et al., 2007). The combined action of RecQ helicases with nucleases is a common feature of this protein family, for example in the RecFOR pathway RecQ works in concert with RecJ (Morimatsu and Kowalczykowski, 2003; Bennett and Keck, 2004). It may be interesting to identify whether Hel308 works in combination with nuclease in this way to process DNA ends.
Each of these models results in a RadA nucleofilament that can initiate recombination or promote reannealing of the parental duplex for NER (figure 5.18). An alternative pathway involves the ‘chicken foot’ intermediate. An additional proposal describes Hel308 catalysing the unwinding of the nascent leading and lagging strands, which would then anneal together. This structure could then be resolved causing the fork to collapse and replication could restart via a D-loop intermediate, analogous to the action of RecG at a blocked fork (McGlynn et al., 2001; McGlynn and Lloyd, 2002; Krejci et al., 2003).
Bacterial UvrD, another 3’-5’ helicase, has been shown to disrupt RecA nucleoprotein filaments, limiting recombination (Veaute et al., 2005). Rather than promoting strand exchange, Hel308 may function in this way to prevent it. RecQ proteins are also known to both promote and inhibit recombination (Bennett and Keck, 2004).
P. furiosus Hel308 was shown to interact directly with RadA, the archaeal RecA (Fujikane et al., 2006). This is a result that compliments either of these suggestions for Hel308.
Figure 5.18. A model for the action of Hel308 at a stalled replication fork
A lesion on the leading strand template causes the replication fork to stall, which results in a gap since the leading strand cannot be primed downstream of the start site. It is thought that Hel308 (red ring) is targeted to the stalled fork, similar to bacterial RecBCD and PriA. The DNA displacement model describes the process by which Hel308 displaces the nascent lagging strand clearing the template for ssDNA binding proteins, for example RadA (euk Rad51, E. coli RecA). The protein displacement model suggests that Hel308 acts in a manner analogous to bacterial RecFOR and displaces the single stranded binding protein SSB (euk RPA), shown as a green sphere, clearing the strand for fork regression or RadA (blue sphere) loading. Both of these models lead to the production of a RadA nucleofilament active in strand exchange. Alternatively, RadA could promote reannealing of the parental duplex for NER. The replication fork is restored by either Holliday junction resolution or end processing of the nascent strands, respectively.
A further model implicates Hel308 in unwinding both the nascent leading and lagging strand to promote the formation of the ‘chicken’ foot intermediate. This is then resolved causing fork collapse, leading to homologous recombination. The replication fork is reset via a D-loop intermediate.