The Nucleus and Gene Expression
Helicases that interact with replication forks: new candidates from archaea E.L. Bolt1 Institute of Genetics, School of Biology, Queen’s Medical Centre, University of Nottingham, Nottingham NG 72UH, U.K.
Abstract Overcoming DNA replication fork blocks is essential for completing genome duplication and cell division. Archaea and eukaryotes drive replication using essentially the same protein machinery. Archaea may be a valuable resource for identifying new helicase components at advancing forks and/or in replication-restart pathways. As described here, these may be relevant to understanding genome instability in metazoans.
Replication fork stalling
Restarting replication in bacteria and yeast
Origins of replication are the starting blocks firing genome duplication in all organisms. At origins, the co-ordinated actions of DNA loading proteins, primases, polymerases and helicases establish a DNA replication machine, the replisome, which drives genome duplication at replication forks at least once before cell division. Problems occur when the replisome becomes compromised at sites remote from origins, circumstances which are currently most clearly defined in bacteria [1]. These flashpoints could arise when the replicative helicase (e.g. bacterial DnaB and archaeal/eukaryotic MCM helicase) cannot separate parental duplex strands, or when processive polymerases (bacterial PolIII, eukaryotic Polδ/Polε and perhaps archaeal PolB) cannot copy template bases (Figure 1A). Potentially, this uncouples replicative polymerase and helicase activities, leading to aberrant DNA structures at forks [2]. A multitude of factors may result in these helicase/polymerase problems, including physical blocks (e.g. transcription complexes) or DNA template strand lesions and breaks. Forks also stall in response to upstream signals that detect ssDNA (single-stranded DNA) or template damage. Whatever the cause of fork demise, replication must eventually restart at these sites away from origins, requiring DNA processing that circumvents or fixes the problem. For example, a route that could overcome template blocks to the leading strand DNA polymerase (Figure 1B) was first proposed in mammalian cells [3] and involves the formation of a Holliday junction ‘chickenfoot’, where nascent strands anneal allowing polymerase template switching (Figure 1B). The summary presented here scratches the surface of how helicases contribute to replication-restart. It then explains a simple in vivo method that led us to investigate the biochemistry of a helicase from archaea that may interact with DNA molecules arising at sites of replication fork stalling. This helicase from archaea, Hel308a, is conserved in higher eukaryotes, including human, but is absent from bacteria and yeasts.
DNA helicases are important for restarting replication in Escherichia coli in multiple pathways, depending largely on the nature of the DNA lesion (i.e. the type of DNA substrate available) generated at stalled forks. For example, different mechanisms may deal with aberrant ssDNA regions present on the lagging strand template compared with the leading strand template [4,5]. Homologous recombination may be important for generating DNA structures that promote replisome reassembly and replication-restart at sites away from the origin. PriA helicase is central to these reactions in bacteria [6]. Recombination prior to replisome reassembly can also direct accurate repair of DNA lesions, processes that are reliant on helicases such as RecG and RuvAB. In eukaryotes too, recombination proteins direct replication restart perhaps by channelling aberrant DNA through BIR (break-induced replication), which may be the eukaryotic equivalent of PriA-directed restart in bacteria [7,8]. Other helicases with important functions for processing DNA structures at stalled forks include UvrD and Rep, and RecQ helicases [9]. Exactly how the known biochemical activities and genetics of these proteins fit into fork recovery is not totally clear in all cases, but each helicase seems to promote genome stability and progression of replication forks.
Key words: archaea, DNA repair, Hel308, helicase, replication fork, replication restart. Abbreviation used: ssDNA, single-stranded DNA. 1 email
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Archaea: new ideas for processing stalled forks in eukaryotes? Archaea, the third domain of life, generally lack orthologues of the above-mentioned helicases. The one exception to this is RecQ, but this is found in only a few species of archaea, all from the genus Methanosarcina. It is likely to represent horizontal gene transfer from bacteria rather than conservation over evolutionary time. RecQ helicases exist in multiple forms in eukaryotes, e.g. Sgs1/Rqh1 in yeasts and WRN, BLM, RecQ4 and RecQ5 in metazoans, where they are crucial for maintaining genome stability [10]. The general absence from archaea of RecQ helicases led us to ponder whether helicases with analogous functions to RecQ exist in archaea. Owing to the similarities between DNA replication and (some) repair pathways in archaea and eukaryotes, we saw this as a potential route to finding helicases, distinct from the C 2005
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Figure 1 Impeding the replisome, and replication restart without
Figure 2 Hel308 from archaea targets model DNA forks and
recombination (A) Cartoon showing possible compromise of a processive replication fork
unwinds lagging strands in preference to the parental strand duplex
through a DNA backbone nick, inter-strand DNA cross-link or DNA base or backbone lesion (black triangle). These can block the replicative helicase (Hel) separating the parental strands, or cause fork collapse directly, or
(A) Helicase action of archaeal Hel308 (Hel308a) (10 nM), unwinding a radiolabelled model Holliday junction substrate (2 nM) as a function of time in 5 mM Mg2+ and 5 mM ATP at 50◦ C. Approximately 20% of
block replicative polymerases (Pol). Uncoupling of helicase and polymerase can lead to formation of aberrant ssDNA. RPA, replication protein A; SSB, single-stranded-DNA-binding protein. (B) One method by
Holliday junction substrate is unwound to flayed duplex DNA in 10 min. (B) The same reaction of Hel308a on a fork with a lagging strand only shows that nearly 100% of substrate is unwound to flayed duplex.
which genome duplication can continue at a leading strand block shown here is through polymerase template switching through interconversion of a fork and a Holliday junction. This may also serve to expose the template lesion for repair.
RecQ family, which may be involved in maintaining genome stability, in archaea and possibly eukaryotes too.
Hel308 from archaea targets replication forks in vivo and in vitro Genetic methods in archaea are advancing rapidly [11], but have not reached the point at which we can search for helicases that interact with stalled forks. As an alternative, I have used a simple heterologous genetics method, inserting archaeal genes into bacterial strains that conditionally stall replication forks. This method seeks a phenotype as a ‘smoking gun’, indicating that an archaeal gene, perhaps a predicted helicase/translocase, may interact with DNA at sites of stalled forks. Crucially, we then aim to test the candidate protein in detail biochemically. Screening genes from hyperthermophilic archaea (or bacteria) in E. coli is problematic, because of large differences in optimal temperatures. However, individual genes from moderately thermophilic C 2005
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archaeal species, such as Methanothermobacter thermautotrophicus, have revealed useful phenotypes in E. coli, leading to biochemical analysis. This organism (M. thermautotrophicus) is also proving useful for screening genomic DNA libraries assembled on to plasmids supported by E. coli, since it has a small (1.8 Mb) and fully sequenced genome. Using the conditional fork-stalling E. coli strain (dnaE486 recQ), developed by Hishida et al. [12], we recently identified that the archaeal orthologue of metazoan Hel308/ Mus308 helicases gives a phenotype in this strain that is indistinguishable from that obtained by reintroducing RecQ [13]. This was indicative of Hel308 interacting with DNA that arises at stalled replication forks. This triggered a detailed biochemical analysis of the recombinant Hel308 protein from Methanothermobacter, to characterize fully its activity on a broad range of DNA substrates in vitro [13]. The most significant features of this were as follows. (i) Minimal ATPase activity and 3 –5 -helicase polarity of archaeal Hel308 (which we call Hel308a, for Hel308archaea) was the same as that reported for human Hel308 [14]. (ii) Hel308a binds to and unwinds with very high efficiency partial fork DNA substrates containing only a lagging strand (i.e. with exposed ssDNA on the leading strand template) (Figure 2). (iii) The helicase action of Hel308a at any fork targets displacement of
The Nucleus and Gene Expression
the lagging strand rather than dissociation of parental duplex or the leading strand. The biochemical data from archaeal Hel308a may begin to offer guidance towards explaining phenotypes observed from a genetic knockout of Hel308 in Drosophila, which shows sensitivity to agents that block DNA replication [15]. To define further the cellular role of Hel308 helicases by biochemistry, it might be instructive to determine interactions, if any, between Hel308a and other proteins of repair and replication. It should also be interesting to dissect the helicase mechanism of Hel308a by introducing defined mutations into the protein and chemical modifications into its substrates. The Wellcome Trust funds this work through a Research Career Development Fellowship award to E.L.B.
2 Byun, T.S., Pacek, M., Yee, M.C., Walter, J.C. and Cimprich, K.A. (2005) Genes Dev. 19, 1040–1052 3 Higgins, N.P., Kato, K. and Strauss, B. (1976) J. Mol. Biol. 101, 417–425 4 Heller, R.C. and Marians, K.J. (2005) Mol. Cell 17, 733–743 5 Michel, B., Grompone, G., Flores, M.J. and Bidnenko, V. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 12783–12788 6 Sandler, S.J. and Marians, K.J. (2000) J. Bacteriol. 182, 9–13 7 Flores-Rozas, H. and Kolodner, R.D. (2000) Trends Biochem. Sci. 25, 196–200 8 Paques, F. and Haber, J.E. (1999) Microbiol. Mol. Biol. Rev. 63, 349–404 9 Cobb, J.A., Bjergbaek, L., Shimada, K., Frei, C. and Gasser, S.M. (2003) EMBO J. 22, 4325–4336 10 Hickson, I.D. (2003) Nat. Rev. Cancer 3, 169–178 11 Allers, T. and Mevarech, M. (2005) Nat. Rev. Genet. 6, 58–73 12 Hishida, T., Han, Y.W., Shibata, T., Kubota, Y., Ishino, Y., Iwasaki, H. and Shinagawa, H. (2004) Genes Dev. 18, 1886–1897 13 Guy, C.P. and Bolt, E.L. (2005) Nucleic Acids Res. 33, 3678–3680 14 Marini, F. and Wood, R. (2001) J. Biol. Chem. 277, 8716–8723 15 Laurencon, A., Orme, C.M., Peters, H.K., Boulton, C.L., Vladar, E.K., Langley, S.A., Bakis, E.P., Harris, D.T., Harris, N.J., Wayson, S.M. et al. (2004) Genetics 167, 217–231
References 1 Cox, M.M., Goodman, M.F., Kreuzer, K.N., Sherratt, D.J., Sandler, S.J. and Marians, K.J. (2000) Nature (London) 404, 37–41
Received 22 June 2005
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