Part I: Structural framework for the mechanism of archaeal exosomes in RNA processing; Part II: Structural insights into DNA duplex separation by the archaeal superfamily 2 helicase Hel308

Part I The exosome is a conserved 3´- 5´ exoribonuclease complex
involved in cellular RNA metabolic processes in eukaryotes and
archaea. Its involvement in the accurate processing of nuclear RNA
precursors and in the degradation of RNA in both nucleus and
cytoplasm implies a central function in the eukaryotic RNA
surveillance machinery. This widespread function implies the
ability of the exosome to distinguish between RNA substrates that
should be matured by the removal of nucleotides to a precisely
defined end point, and defective RNAs that undergo rapid and
complete degradation. However, the structural and molecular
mechanisms of processive 3´- 5´ RNA degradation and substrate
specificity remain unclear. To obtain insights into the structural
and functional organization of the exosome, I determined crystal
structures of two 230 kDa nine subunit exosome isoforms from
Archaeoglobus fulgidus. Both exosome isoforms contain a hexameric
ring of RNase PH-like domain subunits Rrp41 and Rrp42 with a
central chamber. A trimer of Rrp4 or Csl4 subunits is situated on
one side of the RNase PH domain ring and forms a multidomain
macromolecular interaction surface with central S1 domains and
peripheral KH and zinc-ribbon domains. Tungstate soaks identified
three phosphorolytic active sites inside the central processing
chamber. Additional structural and functional results suggest that
the S1 domains of Rrp4 or Csl4 subunits and a subsequent neck in
the RNase PH domain ring form an RNA entry pore that only allows
access of unstructured RNA to the active sites. The structural
results presented here can not only mechanistically unify observed
features of exosomes, including processive 3´ RNA degradation of
unstructured RNA, the requirement for regulatory factors and
coactivators to degrade structured RNA, and the precision in
processing RNA species to a defined length. But the high
conservation of the archaeal exosome to the eukaryotic exosome and
its additional high structural similarity to bacterial
mRNA-degrading PNPase suggest a common basis for 3´ RNA-degradation
in all kingdoms of life. Furthermore, the structure of the archaeal
exosome reveals remarkable architectural and functional
similarities to the protein degrading proteasome. Part II Adenosine
triphosphate (ATP) dependent nucleic acid unwinding by superfamily
2 (SF2) helicases is required for numerous biological processes,
including DNA recombination, RNA decay and viral replication. The
structural and molecular mechanism for processive duplex unwinding
of SF2 helicases is still unclear, in part due to a lack of
structural insights into the actual strand separation reaction.
Archaeal SF2 helicase Hel308 preferentially unwinds lagging strands
at replication forks and is closely sequence related to human PolΘ
and Hel308 as well as Drosophila Mus308. Furthermore, the RecA
ATPase-core of archaeal Hel308 shares high sequence conservation to
the SF2 RNA decay factors Ski2p and Mtr4p. Thus, archaeal Hel308
appears as representative model to understand processive 3´- 5´ DNA
unwinding by SF2 helicases. During this PhD thesis crystal
structures of Archaeogloubs fulgidus Hel308 (afHel308) in the
absence and presence of a 15mer duplex DNA containing a 10mer
3´-overhang were determinded using X-ray crystallography. afHel308
exhibits two typical SF2 RecA-like domains at the N-terminus. The
C-terminus comprises a winged-helix (WH) domain, followed by a
unique seven-helix-bundle domain and a helix-loop-helix (HLH)
domain. The DNA bound structure captures the initial duplex
separation and argues that initial strand separation does not
require ATP binding. Comparison with ATP bound SF2 enzymes suggests
that ATP binding and hydrolysis promotes processive unwinding of
one base pair by a ratchet like transport of the 3’ product strand.
In addition, the structure suggests that unwinding is promoted by a
prominent β-hairpin loop. The identification of similar β-hairpin
loops in Hepatitis C virus (HCV) NS3 helicase and RNA decay factors
Ski2p and Mtr4p, and consistency of the results with biochemical
data on HCV NS3 helicase argue that the observed duplex unwinding
mechanism is applicable to a broader subset of processive SF2
helicases. Furthermore, the interaction between afHel308 and its
DNA substrate also may explain how afHel308 is targeted to branched
nucleic acid substrates.The presented results provide a first
structural framework for duplex unwinding by processive SF2
helicases and reveal important mechanistic differences to SF1
helicases and the SF2 helicase RecG.