Structural and functional analysis of ATP dependent conformational changes in the bacterial Mre11:Rad50 catalytic head complex

The integrity of the genome displays a central role for all living
organisms. Double strand breaks (DSBs) are probably the most
cytotoxic and hazardous type of DNA lesion and are linked to
cancerogenic chromosome aberrations in humans. To maintain genome
stability, cells use various repair mechanisms, including
homologous recombination (HR) and non-homologous end-joining (NHEJ)
pathways. The Mre11:Rad50 (MR) complex plays a crucial role in DSB
repair processes including DSB sensing and processing but also
tethering of DNA ends. The complex consists of the evolutionarily
conserved core of two Rad50 ATPases from which a long coiled-coil
region protrudes and a dimer of the Mre11 nuclease. Even though
various enzymatic and also structural functions of MR(N) could be
determined, so far the molecular interplay of Rad50´s ATPase
together with DNA binding and processing by Mre11 is rather
unclear. The crystal structure of the bacterial MR complex in its
nucleotide free state revealed an elongated conformation with
accessible Mre11 nuclease sites in the center and a Rad50 monomer
on each outer tip, thus suggesting conformational changes upon ATP
and/or DNA binding. However, so far high resolution structures of
MR in its ATP and/or DNA bound state are lacking. The aim of this
work was to understand the ATP-dependent engagement-disengagement
cycle of Rad50´s nucleotide binding domains (NBDs) and thereby the
ATP-controlled interaction between Mre11 and Rad50. For this
purpose high resolution crystal structures of the bacterial
Thermotoga maritima (Tm) MR complex with engaged Rad50 NBDs were
determined. Small angle x-ray scattering proved the conformation of
the nucleotide bound complex in solution. DNA affinity was also
analyzed to investigate MR´s DNA binding mechanism. ATP binding to
TmRad50 induces a large structural change and surprisingly, the NBD
dimer binds directly in the Mre11 DNA binding cleft, thereby
blocking Mre11’s dsDNA binding sites. DNA binding studies show that
MR does not entrap DNA in a ring-like structure and that within the
complex Rad50 likely forms a dsDNA binding site in response to ATP,
while the Mre11 nuclease module retains ssDNA binding ability.
Finally, a possible mechanism for ATP dependent DNA tethering and
DSB processing by MR is proposed.