The HUH Catalytic site
Overall Protein Architecture.
bacteriophage fX174 protein A (gpA) was the first identified
HUH superfamily member (see (Kornberg &
Baker, 1992)) although, surprisingly, no structural information is
related proteins subsequently identified by bioinformatics (Ilyina & Koonin, 1992, Koonin & Ilyina, 1993) included proteins involved in catalysis of viral and plasmid rolling circle replication
(RCR), conjugative plasmid transfer, and DNA transposition (Kapitonov & Jurka, 2001, Garcillan-Barcia, et al., 2002, Ronning, et al., 2005, Ton-Hoang, et al., 2005, Toleman, et al., 2006, Garcillan-Barcia, et al., 2009). They all
carry conserved protein motifs, including the "HUH" motif composed
of two histidine (H) residues separated by a bulky hydrophobic (U) residue, and
the Y-motif containing either one or two tyrosine (Tyr) residues (found in Y1
and Y2 enzymes respectively).
Y1 HUH enzymes (Fig 1.41.1) include Rep
proteins of some plasmids with ssDNA replication intermediates (such as pUB110 (Gruss & Ehrlich, 1989), a wide range of
eukaryotic viruses (Rosario, et al., 2012), most conjugative plasmid relaxases (de la Cruz, et
al., 2010, Guglielmini, et al.,
2011), ISCR (insertion
sequences related to IS91(Toleman, et al., 2006)) and IS200/IS605 insertion
sequence family transposases (Ronning, et al., 2005, Ton-Hoang, et al., 2005). Y2 enzymes
include fX174 gpA itself, Rep proteins of other isometric
ssDNA and dsDNA phages (e.g. phage P2 (Odegrip
& Haggard-Ljungquist, 2001)), some cyanobacterial and archaeal
plasmids and parvoviruses (e.g. adeno-associated virus, AAV) as well as
transposases of the IS91 and helitron
families (Kapitonov & Jurka, 2001),
and MOBF family plasmid relaxases. In some cases, both Y residues
are mechanistically important while for others, only one of the pair appears to
HUH enzymes use a unique mechanism for catalysing ssDNA breakage and
joining. The active site tyrosine creates a 5'-phosphotyrosine intermediate and
a free 3'-OH at the cleavage site (Fig 1.41.1). The
3'-OH can be used for different tasks. The most obvious is to prime
replication, as observed for HUH domains in single-stranded phage Rep proteins,
RCR plasmids and conjugative relaxases. The 3'-OH group can also act as the
nucleophile for strand transfer to resolve the phosphotyrosine intermediate in
the termination step of RCR replication, conjugative transfer and
The HUH enzyme cleavage polarity is inverse to that of the tyrosine
recombinases, which make 3' phosphotyrosine intermediates and generate free
5'-OH groups that cannot be used as replication primers (Grindley, et al., 2006).
HUH enzymes also require a divalent metal ion to facilitate cleavage by
localizing and polarising the scissile phosphodiester bond in contrast to the
cofactor-independent tyrosine recombinases. Depending on the enzyme, Mg2+,
Mn2+ or other divalent metal ions can be used in vitro (Datta, et al., 2003, Larkin, et
al., 2005, Boer, et al., 2006,
Boer, et al., 2009, Hickman, et al., 2010, Edwards, et al., 2013). It is presumed
that Mg2+ or Mn2+ are the physiological cofactors. The
HUH histidine pair provides two of the three ligands necessary for metal ion
coordination (Fig 1.41.1). The
location and identity of the third, invariably polar (Glu, Asp, His or Gln)
residue varies across the superfamily.
structures of several Rep and relaxase HUH domains with and without bound DNA
are available e.g. (Datta, et al., 2003, Guasch, et
al., 2003, Larkin, et al., 2003,
Hickman, et al., 2004, Boer, et al., 2006, Boer, et al., 2009, Messing, et
al., 2012). The order of HUH and Y motifs varies in the primary
sequence: in the Relaxase group, the Y-motif is upstream of the HUH-motif
whereas in the Rep group it is downstream (Fig1.41.1). This
"circular permutation" (Koonin & Ilyina,
1993, Dyda & Hickman, 2003, Guasch,
et al., 2003) changes the domain topology. Nevertheless, the
three-dimensional constellation of active site residues is virtually identical
across the superfamily.
diverse HUH protein functions, it is not surprising that other domains are
often appended to the HUH domain (Fig1.41.1). These are often of unknown function but, ATP dependent helicase, zinc
binding, primase and multimerisation domains are recurring themes (Petit, et al., 1998, Bruand & Ehrlich,
2000, Odegrip, et al., 2000,
Kapitonov & Jurka, 2001, Chang, et
al., 2002, Hickman, et al., 2004,
Clerot & Bernardi, 2006). For example, the ssDNA substrates needed by HUH enzymes can be generated by
a dedicated DNA helicase domain C-terminal to the HUH domain (Im & Muzyczka, 1990, Brister & Muzyczka,
1999, Kapitonov & Jurka, 2001, Clerot & Bernardi, 2006) or
alternatively by recruitment of a host helicase (Petit, et al., 1998, Bruand & Ehrlich,
2000, Odegrip, et al., 2000, Chang, et al., 2002). RCR processes use 3'-5'helicase activity acting
on the template strand to facilitate DNA unwinding at the replication fork
while in conjugation, helicases (as part of the relaxase) are transported into
the recipient cell and track 5' to 3' on the transported ssDNA.
nucleases recognize and bind DNA hairpin structures with cleavage sites located
within the hairpin or in the ssDNA on the 5' or 3' side of the stem. The
crucial role of hairpins has been firmly established in many systems including
plasmid conjugation, eukaryotic viral and plasmid replication and transposition (Orozco &
Hanley-Bowdoin, 1996, Brister & Muzyczka, 2000, Ronning, et al., 2005, Ton-Hoang, et
al., 2005, Boer, et al., 2006,
Messing, et al., 2012, Ton-Hoang, et al., 2012). In other
systems, palindromic sequences that can form DNA hairpins are present near the
probable HUH nuclease cleavage sites [Feschotte, 2001] (del Solar, et
al., 1998). Such hairpins can be formed in vivo under a
number of physiological conditions (see (Bikard, et al., 2011)).
Structural studies revealed that
small DNA hairpins can be recognized in several different ways:
sequence-specific recognition of the dsDNA stem; structure-specific recognition
of irregularities in the stem; or sequence-specific recognition of the hairpin
et al., 2003, Hickman, et al.,
2004, Ronning, et al., 2005, Hickman, et al., 2010, Messing, et al., 2012, Edwards, et al., 2013).
The hairpin-flanking DNA - in
many cases in single-stranded form - is also often important for recognition.
Relaxases, for example, make extensive contacts with the bases extending
between the hairpin and cleavage site (Guasch, et al., 2003, Larkin, et al., 2003, Edwards, et al., 2013), and for a
relative of IS200/IS605 transposases, TnpAREP,
nucleotides on the 5' side of the hairpin are crucial for binding and
sequence-specific recognition (Messing, et al., 2012). Other family
members (Hickman, et al., 2004, Ruiz-Maso, et
al., 2007), have more complex binding modes.
HUH enzymes as transposases
Transposases of members of the IS200/IS605(Ton-Hoang, et al.,
2005), IS91 (Mendiola, et al., 1994) and ISCR (Toleman, et al.,
2006) insertion sequence families and the
eukaryotic helitrons (Kapitonov &
Jurka, 2001) are also HUH enzymes. Those
IS200/IS605 family are the best understood.
IS200/IS605 family transposases are single domain proteins with only the essential HUH motif and a single
catalytic Tyr (Y1 transposases, Fig 1.41.1). Both TnpAIS608 and TnpAISDra2 are obligatory dimers and the
active sites are believed to adopt two functionally important conformations,
one in which each is composed of the HUH motif from one monomer and the Tyr
residue carried by an alpha-helix (aD) from
the other (trans configuration), and
the other in which both motifs are contributed by the same monomer (cis configuration). Only the former has
been observed crystallographically.
Similar proteins are
sometimes found associated with repeated Extragenic Palindromes (REP sequences
whose hairpin structures resemble the ends of IS200/IS605 family members.
RCR transposons: IS91, ISCR and Helitron families
The earliest identified HUH domain transposases were those of the IS91 family (Garcillan-Barcia, et al., 2002) and are significantly larger than Y1
transposases (Fig 1.41.1), carry
a Y2 motif and include an N-terminal zinc binding motif and additional domains
of yet unidentified function.
A group of related elements, the ISCRs
often associated with a variety of antibiotic resistance genes (see (Toleman, et al.,
2006)) carry an orf (the CR or
common region) resembling IS91 family transposases but
with only a single Tyr (Fig 1.41.1). In
addition, eukaryotic relatives, the Helitrons, have been identified by
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