sign in sign up
<<
Home , DNA ligase, DNA polymerase, Primase

Review TRENDS in Vol.21 No.10 October 2005

The promiscuous

Si-houy Lao-Sirieix, Luca Pellegrini and Stephen D. Bell

MRC Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge, UK, CB2 2XZ

DNA are essential for the initiation of DNA different properties: the DNA activity was replication and progression of the replication fork. substantially reduced and the RNA synthesis significantly Recent phylogenetic analyses coupled with biochemical stimulated [2]. An intriguing implication of this obser- and structural studies have revealed that the arrange- vation is that the large subunit might have the ability ment of catalytic residues within the archaeal and to modulate both and substrate choice of eukaryotic primase has significant similarity to those the catalytic subunit. The mechanism by which this of the Pol X family of DNA-repair . Further- influence is exerted is currently unknown. The highly more, two additional groups of , the / promiscuous nature of the archaeal primase is not primase of the bacterial nonhomologous end-joining restricted to the Pyrococcus ; recent studies have machinery and a putative replicase from an archaeal revealed that the primase from the highly diverged have shown striking functional and structural archaeon Sulfolobus solfataricus also has the ability to similarities to the core primase. The promiscuous nature initiate and extend both RNA and DNA chains for up to of the archaeal primases suggests that these 1 kb or 7 kb, respectively [4]. might have additional roles in DNA repair in the . In , the core primase synthesizes a short oligoribonucleotide primer (between 6 and 15 nt long, Subunit architecture of archaeal and eukaryotic depending on the species studied), which is then extended primases by DNA synthesized by the Pol a component of the Pol DNA primase has a pivotal role in DNA synthesis by a–primase complex [1]. In this light, it is tempting to making a short oligoribonucleotide that acts as a primer speculate that the dual RNA and DNA synthesis for DNA polymerase. Thus, the primase is essential for capabilities of archaeal primases could fulfil primer both leading and lagging strand synthesis. In principle the synthesis and DNA extension functions, respectively. primase needs to act once only on the leading strand, but is However, there is currently no evidence for this hypoth- required for the initiation of every Okazaki fragment on esis. Indeed, experiments from the Ishino laboratory the lagging strand. The DNA primase of eukaryotes and indicate that primers synthesized by archaeal primase archaea is distinct in subunit composition, sequence and are extended by the replicative polymerase in a recon- structure from the bacterial analogue, DnaG [1].In stituted in vitro system [2]. Although archaeal primase contrast to monomeric DnaG, archaeal primases have has the capacity to make DNA primers in vitro, two pieces two subunits [2], and in eukaryotes homologues of these of evidence cast doubt on the in vivo significance of the two subunits further interact with two additional com- property. First, it has been established that archaeal ponents to form the Pol a/primase assembly [1]. In this have RNA at their 50 end [5], and review, we shall refer to the two subunits shared by second, measurement of the apparent Km of the Sulfolobus archaea and eukaryotes as the core primase and refer to primase for nucleoside triphosphates (NTPs) and deoxy- the tetrameric eukaryotic assembly of core primase in nucleoside triphosphates (dNTPs) reveals that the affinity complex with the B subunit and Pol a as the Pol a/primase for dNTPs is at least three orders of magnitude lower than (Table 1). The catalytic activity of the primase lies in the that for NTPs [4]. In addition to possessing primase and small subunit of the core primase [1]. Although the polymerase activities, the Sulfolobus enzyme has recently monomeric single catalytic subunit can be purified in been demonstrated to have 30 nucleotidyl terminal recombinant form, interaction with the larger core activity [4,6]. primase subunits appears to both stabilize the catalytic subunit and also modulate its activity to some degree. Table 1. The subunit architecture of primase and primase/pola a Perhaps the most dramatic example of this has come from from representative archaea and eukaryotes studies of the primase of Pyrococcus species. Initial Eukaryotes Archaea studies of this hyperthermophilic enzyme focused on the Human S. cerevisiae Sulfolobus Pyrococcus Pol a p165 Pol1 –– catalytic subunit in isolation [3]. Remarkably, this enzyme (180 kDa) was capable of synthesizing long (up to 6 kb) DNA strands B subunit p77 Pol12 –– in the absence of , that is, it could both (79 kDa) initiate and extend DNA chains. The isolated enzyme had Primase p50 Pri1 (48 kDa) PriS (38 kDa) Pfup41 little or no ability to synthesize RNA. However, when the small (41 kDa) Primase p59 Pri2 (58 kDa) PriL (36 kDa) Pfup46 heterodimeric enzyme was reconstituted it showed very large (46 kDa) a Corresponding author: Bell, S.D. ([email protected]). The names of the encoding the subunits are listed and the size of the Available online 10 August 2005 products are indicated. www.sciencedirect.com 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.07.010 Review TRENDS in Genetics Vol.21 No.10 October 2005 569

Structural studies of archaeal primases , involving two divalent metal ions, proposed for To date, crystal structures for the isolated catalytic other DNA and RNA polymerases [10]. subunit of archaeal primases from P. horikoshii and The position of the was confirmed by diffu- P. furiosus have been reported [7,8]. The crystallographic sion of uridine 50-triphosphate (UTP) in the P. horikoshii analysis showed that the catalytic subunit folds in a novel primase crystals [8]. The current model of primase tertiary structure made up of two domains: a larger, mixed catalysis postulates the existence of a rate-determining a/b domain, where the catalytic activity resides (prim step when the enzyme is simultaneously bound to two domain), and a smaller a-helical domain of unknown [1]. However, the structure of the primase– function (Figure 1). Inserted onto the fold of the catalytic UTP complex provided no insight into the putative second domain is a -binding motif, which represents a -binding event, which might therefore depend conserved feature of archaeal and eukaryotic primases. on the presence of the DNA template, or on the processing The functional role of the zinc-binding motif is not yet of the first nucleotide. known. Intriguingly, bacterial primases also contain a zinc-binding domain that has been implicated in binding Relatives of primase single-stranded DNA [1,9]. It is, therefore, tempting to The family of archaeal/eukaryotic primase-related mol- speculate that the zinc-binding motif in archaeal and ecules has recently been extended with the characteriz- eukaryotic primases might have an analogous role. How- ation of the ORF904 product of the pRN1 plasmid from ever, it should be noted that, in contrast to the situation the hyperthermophilic archaeon Sulfolobus islandicus with the archaeal primase, the bacterial primase zinc- (Figure 2a). This large protein has multiple functional lies in a domain distinct from the catalytic domains and possesses ATPase, primase and polymerase site. activity [12]. The primase and polymerase activities are Although the archaeal/eukaryotic primase catalytic found in the N-terminal domain of the protein and the subunit fold is novel, some features of the active site, as ATPase activity is associated with a putative gleaned from the available structural data, suggest that domain in the C-terminal region of ORF904. Initial the mode of catalysis is likely to be similar to the general studies classified ORF904 as a member of a novel family mechanism of enzymatic synthesis of both RNA and DNA of DNA polymerases, family E. Interestingly, the bio- proposed earlier [10]. Thus, a triad of aspartate residues, chemical properties of ORF904 are reminiscent of the necessary for catalytic activity and invariant across catalytic subunit of archaeal primase. More specifically, archaeal and eukaryotic primases, can be superimposed the primase can initiate both RNA- and DNA-strand on the catalytic aspartates of the human DNA polymerase synthesis, with a preference for DNA, and in the presence b [11], a member of the X family of polymerases (Figure 1 of dNTPs can extend DNA strands for several kilobases and below). It therefore appears that convergent evolution [12]. Although the ORF904 primase/polymerase domain has driven primases to adopt the same mechanism of shows little primary sequence homology with other nucleic

(a) (b)

α-helical domain Zn Active site

C

Prim N domain

TRENDS in Genetics

Figure 1. The catalytic subunit of the archaeal primase. (a) A cartoon representation of the crystallographic model for the catalytic subunit of the primase [ (PDB) id: 1G71; http://www.rcsb.org/pdb]. Secondary structure elements are colour-coded (a helix is red and b strand is green). The position of the Zn ion is indicated as a blue sphere. The catalytic (prim) and a–helical domains are indicated by brackets. (b) Superposition of active site residues for the P. horikoshii primase (Asp95, Asp97 and Asp280) (PDB id: 1V33) and human DNA polymerase b (Asp190, Asp192 and Asp256) (PDB id: 2BPF). The side chains of the catalytic aspartic acid residues are shown as sticks, and the protein backbone as a ribbon (the primase is coloured in cyan and the pol b in green). The dideoxycytidine 50-triphosphate present in the pol b active site is also shown. www.sciencedirect.com 570 Review TRENDS in Genetics Vol.21 No.10 October 2005

(a) Primase MCM B. cereus primase-MCM Primase SF-3 helicase Primase DnaB R. baltica primase and DnaB S. islandicus ORF904 Primase RTase B. bacteriovorus primase-RT (b) TRENDS in Genetics

Figure 3. Novel primase-related proteins encoded in potentially mobile genetic Zn elements in . In Bacillus cereus and Bdellovibrio bacteriovorus, the primase domain (blue) is fused to a MCM-related helicase domain and domain. In Rhodopirellula baltica, the primase is encoded immediately upstream of a encoding a phage DnaB-type helicase.

Zn chromosome maintenance (MCM) presumptive replicative helicase [14]. Although the primase–MCM fusion is unique to this particular phage, it is interesting to note that the primase-like domain is found in several other contexts in other bacterial . More specifically, as Pyrococcus primase ORF 904 primase domain shown in Figure 3, Rhodopirellula baltica encodes an open

TRENDS in Genetics reading frame (ORF) with high similarity to the B. cereus primase domain (BLAST score of 2!eK9). In R. baltica, Figure 2. The ORF904 primase/helicase from Sulfolobus islandicus pRN1. (a) An this ORF encodes only the primase . However, the illustration of the domain organization of ORF904, with regions homologous to primase and the superfamily 3 (SF-3) indicated. (b) Comparison of the gene encoding the primase lies immediately upstream of positions of the zinc-binding site in Pyrococcus horikoshii (left, PDB 1V33) and one of two R. baltica homologues of the helicase DnaB. ORF904 primase (right, PDB 1RNI). The figures were produced using Pymol (http:// Furthermore, this dnaB homologue appears to be related www.pymol.org). The Zn ion is shown in blue and the catalytic residues shown in red. For clarity, residues 176–277 of P. horikoshii primase are not shown and are to the subfamily of phage DnaB-like helicases (Figure 3). instead indicated by the green dashed line. Additionally, in Bdellovibrio bacteriovorus HD100, a homologue of the B. cereus phage primase domain is found acid polymerizing enzymes, the recent elucidation of the in the N-terminal domain of a large ORF that has crystal structure of this domain of ORF904 has revealed homology to bacterial retron reverse transcriptases. striking structural parallels with the architecture of the With the ever-increasing number of bacterial catalytic centre of the Pyrococcus archaeal primase [7,13].In sequences it will be interesting to determine how particular, the arrangement of metal coordinating acidic ubiquitous the primase domain is. The fact that thus far residues shows tight conservation within a region of b sheet. all three homologues appear associated with mobile Both ORF904 and Pyrococcus enzymes have zinc-binding genetic elements or phage strongly implies that this structural elements adjacent to the active centre of the could be a broadly distributed molecule. enzyme. Surprisingly, however, these motifs are found in unrelated positions in the two enzymes (Figure 2b). Although it has been demonstrated that a positively charged The primase–Pol X connection cleft adjacent to the zinc-binding stem in ORF904 is The first hint that there could be an architectural important for template binding [13], it is not known what relationship between the Pol X family of DNA polymerases function the Pyrococcus zinc-binding motif performs. and primases emerged from sequence comparisons of a However, this observation suggests that ORF904 and range of eukaryotic DNA Pol Xs and the small subunit of archaeal/eukaryotic primase are derived from a common archaeal and eukaryotic primases [15]. With the elucida- ancestor, and further implies that this common ancestor tion of the structure of human DNA Pol b and the first lacked a zinc-binding domain and that two separate crystal structure of an archaeal primase, it became events have occurred and been selected for during apparent that the primary sequence similarity was the evolution of the ORF904 and primase families. mirrored by a degree of structural similarity [7,11]. More specifically, as mentioned above, the positioning of the highly conserved catalytic aspartate residues of archaeal Bacterial mobile genetic elements encoding primase was superimposable on the catalytic core of DNA archaeal/eukaryotic primase proteins Pol b. Interestingly, however, the secondary structural The T7 encodes a protein that has an contexts in which the aspartates are found are dissimilar N-terminal domain related to the bacterial primase DnaG between primase and DNA Pol b, suggesting convergent and a C-terminal helicase domain related to the bacterial evolution. Because these aspartates are also structurally replicative helicase DnaB [1]. A recent bioinformatics conserved among polymerases and are implicated in the study has described the identification of a novel primase- two-metal-ion mechanism of DNA polymerization [10], helicase molecule found in an integrated phage in the Kirk and Kuchta suggested that the archaeal primase bacterium Bacillus cereus (Figure 3). Remarkably, the might possess similar catalytic mechanisms to the family primase and helicase domains show homology not to X polymerases [15]. bacterial DnaG and DnaB, but rather to the archaeal and To date, five Pol X family members have been described eukaryotic primase catalytic subunit and the mini in mammalian cells: Pol b, Pol l, Pol m, Pol s and terminal www.sciencedirect.com Review TRENDS in Genetics Vol.21 No.10 October 2005 571 deoxynucleotidyl transferase (TdT) [16]. These enzymes significant in the context of the broad range of damaged have various roles in DNA replication, repair and recom- DNA substrates that are dealt with by the repair bination processes [17]. Pol b, the reference member of the polymerases. Interestingly, the archaeal primases appear Pol X family, is a template-dependent DNA polymerase to be capable of a broader spectrum of activities than their that also possesses a 50-deoxyribose 50-phosphate (dRP) eukaryotic primase counterparts. activity. The principal role for Pol b is in base In light of the absence of members of the DNA-Pol-X excision repair (BER). TdT has a 22% amino acid identity family in archaeal species (with the exception of a single with Pol b. It is a template-independent DNA and RNA archaeal species, Methanobacterium thermoautotrophicum), polymerase that has a key role in the generation of it is tempting to speculate that the archaeal primases diversity by the V(D)J recombination process. In might have dual functionality, working both as bona fide pre-T-cell extracts, TdT associates with Ku, a hetero- primases but also playing roles in DNA-repair processes. dimeric protein required for V(D)J recombination and for Whether the relative efficiencies of the archaeal primases nonhomologous end joining (NHEJ), suggesting a role for in DNA and RNA synthesis could be modulated by TdT in double-strand break (DSB) repair [18].More interaction with as yet unidentified factors remains an recently, two unusual novel polymerases were added to open question. If the archaeal primases also act as repair the Pol X family: Pol l, which has a high amino acid polymerases, then it is tempting to speculate that as identity (32%) to Pol b [19,20] and Pol m, which is more eukaryotic cells evolved, the primase became dedicated to closely related to TdT (41% amino acid identity) [21]. Like DNA replication and novel polymerases (perhaps co-opted the archaeal primases, both enzymes possess template- from mobile genetic elements) adopted specific roles in dependent and template-independent polymerase activi- DNA repair. ties [22,23] and can synthesize DNA de novo [17]. Additionally, these Pol X family members are prone to Acknowledgements frameshift synthesis [24] and can fill in short gaps [19], S-h.L-S. and S.D.B. are funded by the Medical Research Council. L.P. is supporting a role in NHEJ. Finally, Pol l has a dRP lyase funded by the Wellcome Trust. We would like to thank Adam McGeoch for activity used for BER in vitro although its role in this informative discussions. process in vivo is still unclear [25]. References Additional primase-like in DNA repair 1 Frick, D.N. and Richardson, C.C. (2001) DNA primases. Annu. Rev. A further extension to the family of primase-like Biochem. 70, 39–80 molecules has been made with the exciting discovery of a 2 Liu, L.D. et al. (2001) The archaeal DNA primase – biochemical characterization of the p41-p46 complex from Pyrococcus furiosus. pathway for NHEJ in . Initial bioinformatics J. Biol. Chem. 276, 45484–45490 studies by the Koonin, Jackson and Doherty laboratories 3 Bocquier, A.A. et al. (2001) Archaeal primase: bridging the gap revealing the presence of prokaryotic homologues of the between RNA and DNA polymerases. Curr. Biol. 11, 452–456 essential NHEJ factor, Ku [26,27], have been extended 4 Lao-Sirieix, S.H. and Bell, S.D. (2004) The heterodimeric primase of with a series of biochemical and genetic analyses, the hyperthermophilic archaeon Sulfolobus solfataricus possesses DNA and RNA primase, polymerase and 30-terminal nucleotidyl primarily focusing on bacterial organisms encoding this transferase activities. J. Mol. Biol. 344, 1251–1263 pathway [28–30]. In contrast to the highly complicated 5 Matsunaga, F. et al. (2003) Identification of short ‘eukaryotic’ Okazaki eukaryotic NHEJ machinery, the prokaryotic apparatus fragments synthesized from a prokaryotic replication origin. EMBO appears to require just two gene products [28]. One is the Rep. 4, 154–158 6 De Falco, M. et al. (2004) The DNA primase of Sulfolobus solfataricus homologue of Ku, and the other has been named as LigD. is activated by substrates containing a -rich bubble and has a Intriguingly, like the ORF904 protein described above, 30-terminal nucleotidyl-transferase activity. Nucleic Acids Res. 32, LigD is a multifunctional enzyme. In the case of LigD, the 5223–5230 enzyme has ligase, polymerase, and primase acti- 7 Augustin, M.A. et al. (2001) Crystal structure of a DNA-dependent vities [28,30]. Significantly, examination of the sequence of RNA polymerase (DNA primase). Nat. Struct. Biol. 8, 57–61 8 Ito, N. et al. (2003) Crystal structure of the Pyrococcus horikoshii DNA this protein has revealed homology with the archaeal/ primase-UTP complex: implications for the mechanism of primer eukaryotic primase catalytic subunit. Further, biochemical synthesis. Genes Cells 8, 913–923 experiments have shown that, like archaeal primase, LigD 9 Pan, H. and Wigley, D.B. (2000) Structure of the zinc-binding domain can synthesize RNA primers, has DNA-dependent DNA and of Bacillus stearothermophilus DNA primase. Structure 8, 231–239 RNA polymerase activity and, like Sulfolobus primase, has 10 Steitz, T.A. et al. (1994) A unified polymerase mechanism for 0 nonhomologous DNA and RNA polymerases. Science 266, 2022–2025 3 nucleotidyl terminal transferase activity [28,31]. 11 Sawaya, M.R. et al. (1994) Crystal-structure of rat DNA-polymerase- beta – evidence for a common polymerase mechanism. Science 264, Concluding remarks 1930–1935 Thus, the archaeal/eukaryotic primases and a range of 12 Lipps, G. et al. (2003) A novel type of replicative enzyme harbour- repair and mobile genetic element DNA polymerases are ing ATPase, primase and DNA polymerase activity. EMBO J. 22, 2516–2525 clearly related in both the arrangement of the catalytic 13 Lipps, G. et al. (2004) Structure of a bifunctional DNA primase- aspartates within the active centre of the enzymes and by polymerase. Nat. Struct. Mol. Biol. 11, 157–162 a common promiscuity in the reactions they can carry out. 14 McGeoch, A.T. and Bell, S.D. (2005) Eukaryotic/archaeal primase and It appears therefore that this arrangement of the catalytic MCM proteins encoded in a bacteriophage genome. Cell 120, 167–168 15 Kirk, B.W. and Kuchta, R.D. (1999) Arg304 of human DNA primase is site has a degree of functional malleability that is ideally a key contributor to catalysis and NTP binding: primase and the suited to performing nucleic acid synthetic functions on a family X polymerases share significant sequence homology. Biochem- diverse range of substrates. This adaptability could be istry 38, 7727–7736 www.sciencedirect.com 572 Review TRENDS in Genetics Vol.21 No.10 October 2005

16 Hubscher, U. et al. (2002) Eukaryotic DNA polymerases. Annu. Rev. polymerase lambda. Implications for function. J. Biol. Chem. 278, Biochem. 71, 133–163 34685–34690 17 Ramadan, K. et al. (2004) De novo DNA synthesis by human DNA 25 Garcia-Diaz, M. et al. (2001) Identification of an intrinsic 50-deoxyribose- polymerase lambda, DNA polymerase mu and terminal deoxyribo- 5-phosphate lyase activity in human DNA polymerase lambda: a nucleotidyl transferase. J. Mol. Biol. 339, 395–404 possible role in . J. Biol. Chem. 276, 34659–34663 18 Mahajan, K.N. et al. (1999) Association of terminal deoxynucleotidyl 26 Aravind, L. and Koonin, E.V. (2001) Prokaryotic homologs of the transferase with Ku. Proc. Natl. Acad. Sci. U. S. A. 96, 13926–13931 eukaryotic DNA-end-binding protein Ku, novel domains in the Ku 19 Garcia-Diaz, M. et al. (2002) DNA polymerase lambda, a novel DNA protein and prediction of a prokaryotic double-strand break repair repair enzyme in human cells. J. Biol. Chem. 277, 13184–13191 system. Genome Res. 11, 1365–1374 20 Garcia-Diaz, M. et al. (2000) DNA polymerase lambda (Pol lambda), a 27 Doherty, A.J. et al. (2001) Identification of bacterial homologues of the novel eukaryotic DNA polymerase with a potential role in meiosis. Ku DNA repair proteins. FEBS Lett. 500, 186–188 J. Mol. Biol. 301, 851–867 28 Della, M. et al. (2004) Mycobacterial Ku and ligase proteins constitute 21 Dominguez, O. et al. (2000) DNA polymerase mu (Pol mu), homologous a two-component NHEJ repair . Science 306, 683–685 to TdT, could act as a DNA mutator in eukaryotic cells. EMBO J. 19, 29 Weller, G.R. et al. (2002) Identification of a DNA nonhomologous end- 1731–1742 joining complex in bacteria. Science 297, 1686–1689 22 Ruiz, J.F. et al. (2003) Lack of sugar discrimination by human Pol mu 30 Zhu, H. and Shuman, S. (2005) A primer-dependent polymerase requires a single glycine residue. Nucleic Acids Res. 31, 4441–4449 function of Pseudomonas aeruginosa ATP-dependent DNA ligase 23 Ramadan, K. et al. (2003) Human DNA polymerase lambda possesses (LigD). J. Biol. Chem. 280, 418–427 terminal deoxyribonucleotidyl transferase activity and can elongate 31 Gong, C. et al. (2005) Mechanism of nonhomologous end-joining in RNA primers: implications for novel functions. J. Mol. Biol. 328, 63–72 mycobacteria: a low-fidelity repair system driven by Ku, ligase D and 24 Bebenek, K. et al. (2003) The frameshift infidelity of human DNA ligase C. Nat. Struct. Mol. Biol. 12, 304–312

AGORA initiative provides free agriculture journals to developing countries

The Health Internetwork Access to Research Initiative (HINARI) of the WHO has launched a new community scheme with the UN Food and Agriculture Organization.

As part of this enterprise, Elsevier has given 185 journals to Access to Global Online Research in Agriculture (AGORA). More than 100 institutions are now registered for the scheme, which aims to provide developing countries with free access to vital research that will ultimately help increase crop yields and encourage agricultural self-sufficiency.

According to the Africa University in Zimbabwe, AGORA has been welcomed by both students and staff. ‘It has brought a wealth of information to our fingertips’ says Vimbai Hungwe. ‘The information made available goes a long way in helping the learning, teaching and research activities within the University. Given the economic hardships we are going through, it couldn’t have come at a better time.’

For more information visit: http://www.healthinternetwork.net

www.sciencedirect.com