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Biochemical Society Transactions (2009) Volume 37, part 1
Molecular biology of Hel308 helicase in archaea
Isabel L. Woodman and Edward L. Bolt1
School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, U.K.
Abstract
Hel308 is an SF2 (superfamily 2) helicase with clear homologues in metazoans and archaea, but not in fungi or
bacteria. Evidence from biochemistry and genetics implicates Hel308 in remodelling compromised replication
forks. In the last 4 years, significant advances have been made in understanding the biochemistry of archaeal
Hel308, most recently through atomic structures from cren- and eury-archaea. These are good templates for
SF2 helicase function more generally, highlighting co-ordinated actions of accessory domains around RecA
folds. We review the emerging molecular biology of Hel308, drawing together ideas of how it may contribute
to genome stability through the control of recombination, with reference to paradigms developed in bacteria.
DNA helicases in replication, repair and
recombination
Since many stages of replication, repair and recombination
require ssDNA (single-stranded DNA) substrates, helicases
can oblige by separating strands of base-paired dsDNA
(double-stranded DNA). Unwinding parental duplex for
genome duplication at replication forks requires protein
complexes containing helicases gp41 (bacteriophage), DnaB
(bacteria) or MCM (minichromosome maintenance) (eukaryotes and archaea). Accessory helicases, e.g. PriA, Rep,
UvrD, Dda, PcrA and Rrm3, may aid the processivity of
replicative helicases during strife [1–4]. Chemical damage to
nucleotides, both genome-wide and at replication forks, is
accessed for excision repair by helicases of Uvr (bacteria)
[5], XP and Fanc protein complexes (both eukaryotes and
archaea) [6]. Defects in XP and Fanc proteins in humans
manifest as the disorders Fanconi’s anaemia and xeroderma
pigmentosum, which are characterized by a variety of clinical
symptoms caused by genome instability.
Recombination events are linked to excision repair and
replication to deal with lesions using an intact homologous
DNA molecule as a repair template. The onset and early
stages of recombination may be controlled by helicases as
directional motors that depose proteins or DNA strands to
expose ssDNA that stimulates recombination. Alternatively,
such helicase clearance roles may prevent recombination
by disassembling recombination intermediates. These kinds
of event, summarized in Figure 1, have been linked to
bacterial helicases RecQ [7], UvrD [8] and Rep [9], although
the exact roles of these proteins have been elusive. Late in
recombination, branch migration and dissolution reactions
are catalysed by Holliday junction helicases (e.g. RecQ
[10,11], bacterial RuvAB [12] or RecG [13]) to disentangle
synapsed DNA molecules.
Key words: archaeon, Hel308, helicase, recombination, repair, replication.
Abbreviations used: dsDNA, double-stranded DNA; ORF, open reading frame; PCNA,
proliferating-cell nuclear antigen; SF2, superfamily 2; ssDNA, single-stranded DNA.
1
To whom correspondence should be addressed (email [email protected]).
C The
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Authors Journal compilation Where Hel308 sits in this broad church of genome biology
is discussed below. The absence from archaea of sequence
homologues of bacterial recombination helicases made it
tempting to seek analogous roles for Hel308. Evidence
currently points to involvement of Hel308 early on in
recombination or other gene-conversion events that are
linked to blocked replication, perhaps in a role similar to
that of Escherichia coli RecQ or UvrD protein.
Hel308 in archaea
Designation of Hel308 arose from isolation in Drosophila of
mutations that cause hypersensitivity to DNA-cross-linking
reagents [14]. One group of Drosophila mutants (mus308)
mapped to what is now known as PolQ [15], a translesion
polymerase that is active in excision repair [16]. PolQ
has proposed helicase domains (but no reported helicase
activity) that were used to identify novel human helicases implicated in DNA repair, unearthing hel308 [17]. The rapid
accumulation of genome sequence data identified Hel308
homologues in archaea, including in the minimalist genome
of the hyperthermophile Nanoarchaeum equitans, but not
in bacteria or yeast. Conservation of DNA replication and
repair protein machinery in eukaryotes and archaea, but
not in bacteria, is not unusual and has been commented on
several times, e.g. reviewed by Barry and Bell [18].
Hel308 [then called ORF (open reading frame) mth810]
from the archaeon Methanothermobacter thermautotrophicus
was first proposed to be part of a predicted exosome, through
sequence similarity to Ski2 helicases involved in releasing
RNA molecules for degradation [19]. No more was heard
until 2005, when Mth810 (molecular mass of 75 kDa) was
described for its actions at forks and Holliday junctions
from M. thermautotrophicus and Pyrococcus furiosus (ORF
PF0677, 82 kDa) [20,21]. Mth810 was called Hel308a (‘a’ for
archaea) on the basis of sequence conservation with human
Hel308, and identical properties of human Hel308 and
Mth810 in ssDNA-stimulated ATPase activity and 3 →5
polarity of unwinding. The P. furiosus enzyme was called
Hjm (Holliday junction migration) for its ability to unwind
Biochem. Soc. Trans. (2009) 37, 74–78; doi:10.1042/BST0370074
Molecular Biology of Archaea
Figure 1 Hel308 in recombination-dependent DNA replication at blocked replication forks
Hel308 (blue shape) loads on to a blocked lagging strand template as a motor that clears DNA and proteins [e.g. RPA
(replication protein A), light blue ovals]. Although this may provoke recombination from ssDNA, it could also remove stalled
protein complexes that interfere with replication or other events. Hel308 may load RadA recombinase (grey hexagons) on
to ssDNA, triggering recombination by strand exchange. Alternatively, a clearance role of Hel308 may extend to removing
RadA from ssDNA, preventing recombination in favour of an alternative repair strategy. In this anti-recombinogenic role,
Hel308 may stabilize a replication fork, promoting repair by translesion polymerases.
model Holliday junctions in vitro. Despite the divergent
nomenclature of these two archaeal Hel308 homologues,
they show very similar biochemical properties in vitro and, as
explained below, they exhibit RecQ-like genetic interactions
with sites of blocked replication in vivo. For lucidity, the
archaeal helicase will be called Hel308.
Reactions of archaeal Hel308: remodelling
replication forks?
It is not yet possible to superimpose biochemical data from
archaeal Hel308 on to less comprehensive genetic data
from mostly eukaryotic studies. Drosophila and Caenorhabditis elegans hel308 promote repair of DNA cross-links
[22,23], and its inactivation in Drosophila provokes homologous recombination [24]. Genetic analysis in the archaeon
Haloferax volcanii showed hel308 to be an essential gene
(T. Allers, personal communication). However, analysis of archaeal hel308 in bacteria, alongside biochemistry of
the isolated protein, has developed a working hypothesis
that archaeal Hel308 may have very similar functions to
the bacterial SF2 (superfamily 2) helicase RecQ, or the
SF1 helicase UvrD. RecQ and UvrD are not widespread in
archaea, and RQC and HRDC motifs common to RecQ
proteins cannot be detected in Hel308 sequences.
Isolated archaeal Hel308 protein is a ssDNA-stimulated
ATPase that unwinds duplex DNA from a 3 (but not
a 5 ) ssDNA overhang as its minimal substrate [20]. No
activity is detectable on fully base-paired duplex DNA. It
unwinds a variety of branched DNA structures, including
an RNA–DNA hybrid [25], but is most active on forked
molecules, removing the lagging strand of a fully base-paired
model fork [20,26]. This substrate versatility is reminiscent
of bacterial RecQ helicases [7]. The genetic screen identifying
M. thermautotrophicus Hel308 showed recQ-like activity in
replication-impaired E. coli strains [20]. Similar genetic analysis showed that Hel308 did not interact with Holliday junctions, and is therefore more likely to be involved in processing
DNA at early stages of events that remodel blocked replication forks [20]. Ideas of how Hel308 may be involved in these
events are described in Figure 1 and are expanded on below.
Is Hel308 in archaea analogous to E. coli
RecQ or UvrD?
RecQ helicases are a family of proteins that contribute to
genome stability by controlling homologous recombination
at replication forks. E. coli RecQ can remodel replication
forks to initiate recombination, by generating ssDNA for
RecA-catalysed strand exchange [27,28], and it can also
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Biochemical Society Transactions (2009) Volume 37, part 1
Figure 2 Hel308 from M. thermautotrophicus efficiently unwinds
DNA at 50◦ C
M. thermautotrophicus Hel308 efficiently unwinds >100 bp of DNA in
5 mM ATP and 5 mM MgCl2 at 50◦ C. Helicase substrate was prepared
from polymerization of 32 P-end-labelled M13 primer annealed to M13
ssDNA. Lane 1, 95 nt marker DNA; lane 2, boiled substrate; lane 3, control
(no Hel308); lane 4, 100 nM Hel308; lane 5, 100 nM K51L Hel308, walker
A mutant.
process branched recombination intermediates when acting
in concert with topoisomerase III [11,29]. The recQ-like
phenotype of introducing archaeal hel308 into E. coli
replication-deficient strains [20,26] may arise because Hel308
gains access to blocked forks, clearing away obstacles to
generate ssDNA that provokes recombination. Alternatively,
this ‘fork clearance’ activity may also act counter to recombination, by removing D-loops and/or recombinase proteins
in favour of alternative events to overcome blocked forks.
This would be more like the bacterial superfamily 1 helicase
UvrD, which is required for E. coli to survive DNA-crosslinking reagents [30]. UvrD has been proposed to have a forkclearing role [8], and may displace stalled protein complexes
in other contexts away from replication forks. Hel308 is
apparently quite processive (Figure 2), and can displace other
proteins and streptavidin beads from ssDNA molecules [31],
the basis of which is explained by atomic structures of
Hel308–DNA complexes (see below) (Figure 3). Archaeal
Hel308 does not complement the UV-sensitivity of an E. coli
uvrD strain (E.L. Bolt, unpublished work), but the recent
development of more refined E. coli recQ, uvrD, rep, recA and
ruv strain combinations may make it possible to distinguish
between recombinogenic and anti-recombinogenic roles of
archaeal Hel308, albeit in a heterologous system.
C The
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Authors Journal compilation Help from its friends?
It may help to judge the function of a helicase if we know
the proteins with which it interacts. Evidence was recently
presented for Hel308 remodelling a fork into a Holliday junction, and for interaction of Hel308 with the resolvase protein,
Hjc [32]. The experiments imply that a blocked replication
fork could be regressed into a Holliday junction by Hel308,
provoking recombination by recruitment of Hjc to catalyse
Holliday junction resolution, generating DNA ends. This
is reminiscent of a model of replication and recombination
interplay first proposed in eukaryotes [33] and later demonstrated for bacterial RecG [13]. No details are available for the
nature of Hel308–Hjc interaction, the mechanics of how this
results in junction resolution or whether it can be supported
with other non-archaeal resolvases. Under some conditions,
Hjc can cleave replication forks as well as Holliday
junctions in vitro (I.L. Woodman and E.L. Bolt, unpublished
work).
A Pyrococcus Hel308 also reportedly interacts with the
sliding-clamp protein PCNA (proliferating-cell nuclear
antigen) [26]. Little is known about Hel308–PCNA
interaction: no mediating amino acid residues have been
confirmed, and there is no idea of how the interaction affects
protein function. A putative PIP (PCNA-interacting protein)
box sequence motif for PCNA interaction at the Hel308
C-terminus was reported in Pyrococcus Hel308 proteins [26],
but this is not present in any other archaea. Hel308 atomic
structures [25,31], and unwinding data from isolated enzyme
(Figure 2), make it unlikely that PCNA is required to
improve the processivity of helicase unwinding, but a role for
PCNA somehow recruiting Hel308 to forks cannot be ruled
out. There is mention, although no data, of Hel308 interacting
with RadA recombinase [26]. This is intriguing as a potential
mechanism for Hel308 and RadA together promoting
recombination after remodelling of a blocked fork. Overall,
more robust studies are needed to determine physical
and functional interactions of Hel308 and their biological
significance.
Atomic structures of Hel308
Atomic structures of Hel308 from the euryarchaeon Archaeoglobus fulgidus [25] and the crenarchaeon Sulfolobus solfataricus [31] revealed a five-domain protein monomer that surrounds DNA and drives a β-hairpin plough through duplex
to separate strands (Figure 3). The structures represent a significant step forward in closing the gap in knowledge between
mechanisms of superfamily 1 and superfamily 2 helicases.
Apparent processivity of Hel308 on simple in vitro
substrates is readily explained by Hel308 clasping DNA using
all five domains. DNA is bent into a ‘dog-leg’ shape that can
be interpreted, with caution, as being equivalent to a fork
structure that is lacking part of a leading strand, but with
an intact lagging strand. DNA is unwound from an initial
2 bp ATP-independent DNA melting by a β-hairpin loop in
domain 2. Further unwinding is fuelled by ATP binding and
hydrolysis in RecA folds shared between domains 1 and 2,
Molecular Biology of Archaea
Figure 3 Atomic structure of Hel308–DNA complex, and the position of domain 5
(A) Image of A. fulgidus Hel308 bound to 3 -tailed ssDNA, produced from PDB code 2P6R (see also [25,34]). Domain 5
(salmon pink) engages with ssDNA 10–12 nt distal from the point of strand separation at the β-hairpin. (B) Arg644 (Arg649 in
M. thermautotrophicus Hel308) engages ssDNA in addition to several other DNA contacts arising from the peptide backbone.
Mutation of Arg649 in M. thermautotrophicus Hel308 severely curtails ssDNA-stimulated ATPase activity. Arg642 (Arg647 in
M. thermautotrophicus) orientates away from DNA as a salt bridge with the helicase ratchet domain 4. (C) Conserved RAR
(Arg-Ala-Arg) motif at the C-terminal domain 5 of Hel308 proteins. Afu, A. fulgidus; Mth, M. thermautotrophicus.
causing co-ordinated movements of a helicase ratchet generated by domains 2 and 4. A proposed helicase mechanism is
described in detail, and goes some way to unifying observed
actions of SF2 helicases more generally, by comparison with
hepatitis C virus helicase NS3 and RNA helicases Ski2
and Vasa [25]. RecQ helicase structures do not share these
unwinding characteristics, although similar cellular functions
of Hel308 and RecQ could have arisen by convergent
evolution of function from different ancestral genes.
An appealing observation from the A. fulgidus structure
is a fingertip-grip on 3 tailed ssDNA by Hel308 domain 5
(Figures 3A and 3B). Biochemistry on this domain showed the
interaction to be functionally important as a ‘molecular brake’
coupling ssDNA binding to ATPase and unwinding activities
[31,34]. We mutated a triad of arginine residues in Mth
Hel308 domain 5 (R647 XRAR651 ) that is conserved in archaeal
and metazoan Hel308 (Figure 3C) [34]. R649A protein had
dramatically reduced ssDNA-stimulated ATPase activity, but
was unchanged in its basal ATPase activity. ATPase activity of
R647A mutant even in the absence of ssDNA was comparable
with the ssDNA-stimulated activity of wild-type protein.
There was no difference in DNA binding by each mutant
protein compared with wild-type, but fork unwinding was
reduced in each case. The A. fulgidus Hel308 atomic structure
revealed that Arg649 (and Arg651 ) contact ssDNA distal to the
RecA core, but Arg647 and other domain 5 residues contact
the helicase ratchet domain 4 (Figure 3B). We interpreted this
as the engagement of Hel308 domain 5 on ssDNA triggering
productive ATPase activity, leading to helicase unwinding. A
similar proposal arose from removal of domain 5 from crenarchaeal Hel308, in this case with a resulting increase in ATPase
activity and helicase unwinding [31]. In this way, Hel308
could be controlled to respond to regions of ssDNA arising at
compromised replication forks, or elsewhere in the genome. It
may be important that there is a significant difference between
the positioning of domain 5 in structures of A. fulgidus
Hel308 with and without DNA, and we await an ATP-bound
structure for more insight. Armed with structural insights
into Hel308 DNA binding by domain 5, 10–12 nt away from
a dsDNA–ssDNA junction, we are using a variety of fork
substrates to test how they are engaged by the helicase and
how the disposition of the fork affects Hel308 function.
Closing remarks
The cellular role of Hel308 in archaea, and metazoans, is
not clear, but a link to controlling potential recombination
events arising during DNA replication seems likely. Atomic
structures give useful insight into the biochemical function of
archaeal Hel308, but genetic analysis in archaea is required to
flesh out ideas from biochemistry and heterologous genetics
of Hel308. This would be useful to establish this helicase into
epistasis groups, and to screen for recombination defects or
hyper-recombination phenotypes.
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Biochemical Society Transactions (2009) Volume 37, part 1
Funding
We are grateful for funding from the Biotechnology and Biological
Sciences Research Council for a studentship to I.L.W. [grant number
BB/D526602/1IW].
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Received 4 August 2008
doi:10.1042/BST0370074