Rad50.lyx

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\begin_body

\begin_layout Chapter
DNA repair protein Rad50
\end_layout

\begin_layout Section
The Mre11-Rad50-Nbs1(Xrs2) complex—architecture and function
\end_layout

\begin_layout Standard
\begin_inset CommandInset label
LatexCommand label
name "ch:Rad50"

\end_inset


\end_layout

\begin_layout Standard
The genetic material of every cell is constantly under the stress – both
 endogenous and exogenous 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 damaging agents, which may introduce 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 to the chromosome.
 In addition, the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 formation sometimes is even a `physiological` phenomenon—the formation
 of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 is an essential step in immunoglobulin gene rearrangements and meiotic
 chromosome recombination 
\begin_inset CommandInset citation
LatexCommand citep
key "Johnson2021,Zhang2022"
literal "true"

\end_inset

.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 arise from exposure to ionizing radiation, and genotoxic agents, and may
 arise spontaneously while 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 is replicated 
\begin_inset CommandInset citation
LatexCommand citep
key "Tubbs2017"
literal "true"

\end_inset

.
 Because even the single 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 event may be a cause of chromosomal aberration, aneuploidy, tumorigenesis,
 and cell death, cells have evolved to sense, signal and repair 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex is crucial for 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 repair as the complex is involved in every step of repair: sensing, 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 end binding, signaling and finally repairing.
 Those processes follow two general routes: 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nhej}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hr}
\end_layout

\end_inset

.
 In the Eukarya the use of one of two pathways is dependent on the cell
 cycle 
\begin_inset CommandInset citation
LatexCommand citep
key "Scully2019"
literal "true"

\end_inset

.
 
\end_layout

\begin_layout Standard
The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

, also known as the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glslink{mre}{Mre11}
\end_layout

\end_inset

 complex is a complex composed of three types of proteins: 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

, 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 or 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{xrs}
\end_layout

\end_inset

.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 is essential for the viability of vertebrate cells (the yeast cells are
 viable without the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

).
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 is engaged in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 metabolism, from which the most important is the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 repair.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 orthologs have been found in every domain of life – which underlines the
 importance of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 
\begin_inset CommandInset citation
LatexCommand citep
key "Pal2020"
literal "true"

\end_inset

.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 (and its homologs) seems to be unique to Eukarya, as no homologs of the
 protein were found in the other domains.
 The X in the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 stands for the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{xrs}
\end_layout

\end_inset

 protein characteristic of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

, sharing a similar function to 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 despite the weak homology.
\end_layout

\begin_layout Standard
Recent five years of 
\begin_inset ERT
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\begin_layout Plain Layout


\backslash
gls{mrn}
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 research showed mechanical and structural insight into the modes of action
 of 
\begin_inset ERT
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\backslash
gls{mrn}
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\end_inset

.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex binds to the homoduplex 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 and scans past the nucleosomes to locate 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 ends.
 The recognition of free 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 ends is inherent to the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

, however,  the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 sliding and nucleosome bypassing is inherent to the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

.
 This function separation may explain why two of the subunits are conserved
 in all domains of life as the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 core complex.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 performs the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 end recognition via facilitated one-dimensional diffusion.
 The sliding (not alternate binding and dissociation) is facilitated by
 the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 subunit.
 Though 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 binds and hydrolyzes 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

, the diffusion coefficients are 
\begin_inset ERT
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\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 independent, which means that 
\begin_inset ERT
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\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 hydrolysis does not regulate facilitated diffusion.
 The region responsible for the one-dimensional facilitated diffusion is
 located along the globular domains of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 is not responsible for the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

-
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 interaction 
\begin_inset CommandInset citation
LatexCommand citep
key "Myler2017"
literal "true"

\end_inset

.
 Due to the unique 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 properties, the whole 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex is able to diffuse on euchromatin and nucleosome-free regions,
 however,  the diffusion on heterochromatin may be hampered, which may be
 the reason why the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hr}
\end_layout

\end_inset

 is limited or the repair of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 at such regions is slow.
\end_layout

\begin_layout Standard
Often the free 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 end created by a 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 event will be blocked by a protein, e.g.
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ku}
\end_layout

\end_inset

.
 In yeast the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 deficient cells are highly sensitive to 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

, this phenotype is attenuated by deletion of the 
\emph on

\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gshort{ku}
\end_layout

\end_inset


\emph default
 gene — the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 has to remove 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ku}
\end_layout

\end_inset

–blocked 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 to enable the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 resistance.
 However,  it should be noted that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ku}
\end_layout

\end_inset

 is not abundant in lower eukaryotes as it is in higher eukaryotes, and
 in most cases in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

 the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 end processing is not required when the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 are not obstructed 
\begin_inset CommandInset citation
LatexCommand citep
key "Llorente2004,Mimitou2008,Zhu2008"
literal "true"

\end_inset

.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ku}
\end_layout

\end_inset


\begin_inset Marginal
status collapsed

\begin_layout Plain Layout
In prokaryotes the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ku}
\end_layout

\end_inset

 is homomeric.
\end_layout

\end_inset

 heterodimer is an abundant protein (especially in human cells) 
\begin_inset CommandInset citation
LatexCommand citep
key "Beck2011"
literal "true"

\end_inset

, combined with the high affinity towards the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 and the recruitment within seconds 
\emph on
in vivo
\emph default
 
\begin_inset CommandInset citation
LatexCommand citep
key "Mari2006"
literal "true"

\end_inset

, the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 (and some other 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

–interacting factors) is essential in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 processing in high eukaryotes 
\begin_inset CommandInset citation
LatexCommand citep
key "Sartori2007"
literal "true"

\end_inset

.
 The process is most likely facilitated by the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 resection and subsequent 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 repair, this is often done by the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 end resection.
\end_layout

\begin_layout Standard
Because the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 can form hairpins or be modified by chemicals, associated proteins (like
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ku}
\end_layout

\end_inset

), or covalently bound proteins 
\begin_inset CommandInset citation
LatexCommand citep
key "Morimoto2019,Johnson2021"
literal "true"

\end_inset

, the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 repair requires the cut.
 The enzymatic cleavage is done by 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

, an Mn(II) dependent metalloenzyme 
\begin_inset CommandInset citation
LatexCommand citep
key "Paull1998,Trujillo1998,Usui1998"
literal "true"

\end_inset

, but how it is done if the 
\emph on
recombinant 
\emph default

\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 
\emph on
in vitro
\emph default
 is a 3'
\begin_inset Formula $\rightarrow$
\end_inset

5' exonuclease? The strict polarity of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 nuclease is counterintuitive, as for the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hr}
\end_layout

\end_inset

 the 5'-
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ssdna}
\end_layout

\end_inset

 strand is required 
\begin_inset CommandInset citation
LatexCommand citep
key "Ranjha2018"
literal "true"

\end_inset

.
 The paradox was fueled by the genetic experiments performed on 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

 showing that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 is responsible for the 5'
\begin_inset Formula $\rightarrow$
\end_inset

3' resection 
\emph on
in vivo
\emph default
.
 How it is possible that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 contributes to the 5' strand degradation, and how the 3'
\begin_inset Formula $\rightarrow$
\end_inset

5' exonucleolytic activity is inhibited? The second question is answered
 by the formation of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 core complex.
 In the cell, the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-loaded 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 inhibits sterically the 3'
\begin_inset Formula $\rightarrow$
\end_inset

5' exonuclease activity.
 Though the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

ase activity of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 is low, in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

 the core 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 complex endonucleolytic–exonucleolytic balance is regulated by 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 hydrolysis.
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
Gls{atp}
\end_layout

\end_inset

 promotes endonucleolytic activity, while 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 hydrolysis switches those activities 
\begin_inset CommandInset citation
LatexCommand citep
key "Majka2012"
literal "true"

\end_inset

.
 
\end_layout

\begin_layout Standard
In Eukarya (and especially in mammals or humans) the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 (or 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{xrs}
\end_layout

\end_inset

 in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

) is much more important in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 in the core 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 complex functioning.
 First of all the 
\begin_inset ERT
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gls{nbs}
\end_layout

\end_inset

(
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{xrs}
\end_layout

\end_inset

) is required for nuclear transport, however,   the nuclease regulation
 differs between 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

.
 In 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

, 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{xrs}
\end_layout

\end_inset

 is not necessary for the endonucleolytic activity of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

, however,   it exerts some positive stimulation on the process, thus in
 the cells with deletion of 
\emph on

\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{xrs}
\end_layout

\end_inset


\emph default
, and nuclear localization signal placed on 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset


\emph on
 
\emph default
the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 end resection was only minimally impaired.
 In 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 is crucial for endonucleolytic activity, which is stimulated by additional
 factors, especially the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ctip}
\end_layout

\end_inset

.
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
Gls{ctip}
\end_layout

\end_inset

 (and in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

, the ortholog of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ctip}
\end_layout

\end_inset

—
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sae}
\end_layout

\end_inset

) stimulates the endonucleolytic nick of 5' 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

, facilitating the formation of the 3' overhanging 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ssdna}
\end_layout

\end_inset

.
\end_layout

\begin_layout Standard
The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 performs this by the cut at the 5' strand in the near vicinity of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 end, followed by exonucleolytic resection from 3'
\begin_inset Formula $\rightarrow$
\end_inset

5' direction 
\begin_inset CommandInset citation
LatexCommand citep
key "Garcia2011,Deshpande2016"
literal "true"

\end_inset

.
\begin_inset Marginal
status open

\begin_layout Plain Layout
to jest znane jako jakiś model czy coś 
\begin_inset Note Note
status open

\begin_layout Plain Layout
C:/Users/makro/Downloads/1-s2.0-S1568786420301178-main.pdf
\end_layout

\end_inset

 może jakiś obrazek?
\end_layout

\end_inset

 The result of this nucleolytic cleavage is 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ssdna}
\end_layout

\end_inset

, an intermediate to be utilized in strand annealing in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nhej}
\end_layout

\end_inset

 or to be further elongated by a 5'
\begin_inset Formula $\rightarrow$
\end_inset

3' degradation in the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hr}
\end_layout

\end_inset

.
 Both 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 and bacterial 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 can enzymatically cleave off the second strand, which results in the release
 of the blocked 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 termini 
\begin_inset CommandInset citation
LatexCommand citep
key "Deshpande2016,Connelly2003"
literal "true"

\end_inset

.
 
\end_layout

\begin_layout Standard
The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 polarity paradox is thus explained by the:
\end_layout

\begin_layout aEnumerate (ClassicThesis)
proteins that bind free 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 ends, block sterically the 3'
\begin_inset Formula $\rightarrow$
\end_inset

5' exonucleolytic activity of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

,
\end_layout

\begin_layout aEnumerate (ClassicThesis)
tight interaction with 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, inhibiting the 3'
\begin_inset Formula $\rightarrow$
\end_inset

5' exonucleolytic activity while 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

–loaded,
\end_layout

\begin_layout aEnumerate (ClassicThesis)
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ctip}
\end_layout

\end_inset

(
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sae}
\end_layout

\end_inset

) and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

(
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{xrs}
\end_layout

\end_inset

) stimulation of the 5'-en
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
-
\end_layout

\end_inset

donu
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
-
\end_layout

\end_inset

cle
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
-
\end_layout

\end_inset

olytic activity of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

.
\end_layout

\begin_layout Section
Globular domain of MRN(X)—the catalytic core
\end_layout

\begin_layout Standard
The globular domain of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex harbors the active centers of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

.
 The globular domain allows association and sliding on the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 in search of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glspl{dsb}
\end_layout

\end_inset

 
\begin_inset CommandInset citation
LatexCommand citep
key "Myler2017"
literal "true"

\end_inset

.
 The globular domain of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 contains the Walker A and B motifs, which do bind 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

.
 The mutation that prevents the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 binding in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{sc}
\end_layout

\end_inset

 is equivalent to 
\begin_inset Marginal
status collapsed

\begin_layout Plain Layout
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
textDelta
\backslash

\end_layout

\end_inset

 before the gene's name indicates deletion.
 
\end_layout

\end_inset


\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
textDelta
\end_layout

\end_inset


\emph on
rad50
\emph default
, however,   the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex with 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 that does not bind 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 the complex was only not functional in the cutting of the 3' overhanging
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ssdna}
\end_layout

\end_inset

 
\begin_inset CommandInset citation
LatexCommand citep
key "Moncalian2004"
literal "true"

\end_inset

.
 The biochemical studies of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 show that 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 hydrolysis by 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 simulates the nucleolytic activity of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 on 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsdna}
\end_layout

\end_inset

 
\begin_inset CommandInset citation
LatexCommand citep
key "Connelly1997,Deshpande2017,Herdendorf2011,Hopfner2000,Paull1999,Trujillo2001"
literal "true"

\end_inset

, while the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 core complex, is active on 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ssdna}
\end_layout

\end_inset

 even in the absence of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 
\begin_inset CommandInset citation
LatexCommand citep
key "Connelly1996,Herdendorf2011,Hopfner2000"
literal "true"

\end_inset

.
 The exact step-by-step catalysis (and associated conformational changes)
 of the nucleolytic activity of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 or 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 is unknown yet.
 Many structures of the catalytic cores of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

/
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 were published to date 
\begin_inset CommandInset citation
LatexCommand citep
key "Hopfner2000a,Hopfner2001,Lammens2011,Lim2011,Liu2016,Mockel2012,Park2011,Seifert2016,Williams2008,Williams2011,Kashammer2019,Rotheneder2023"
literal "true"

\end_inset

, however,  no structural evidence of free 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 ends is shown, in which 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 is bound 
\emph on
after
\emph default
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 hydrolysis.
 Moreover, the structural differences between the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 globular domain from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 and bacterial and archeal 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complexes suggest differences in the mechanism of action 
\begin_inset CommandInset citation
LatexCommand citep
key "Park2011"
literal "true"

\end_inset

.
\end_layout

\begin_layout Standard
Till this year (2023) the structural detail of the interaction of human
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 with the catalytic core of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 was unknown.
 Recent advances in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cryoem}
\end_layout

\end_inset

 allowed for the visualization of the interaction of the human 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 with the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 core complex.
 
\begin_inset CommandInset citation
LatexCommand citep
key "Rotheneder2023"
literal "true"

\end_inset

 Contrary to previous assumptions (and structures), 
\emph on
single
\emph default
, not two 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 particles wrap around the dimeric 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

.
 The binding is centered, and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 wraps the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
glslink{mr}{
\backslash
ce{M2R2}}
\end_layout

\end_inset

 in a pseudo-symmetric fashion, the feature seems to be evolutionarily conserved
 across various eukaryotes.
 
\begin_inset CommandInset citation
LatexCommand citep
key "Rotheneder2023"
literal "true"

\end_inset


\end_layout

\begin_layout Standard
The dynamicity of the complex differs between the domains, and the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 complex behavior from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ec}
\end_layout

\end_inset


\begin_inset Marginal
status open

\begin_layout Plain Layout
The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 complex in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ec}
\end_layout

\end_inset

 is called SbcC–SbcD complex.
\end_layout

\end_inset

 differs from the eukaryotic 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 or 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cthermo}
\end_layout

\end_inset

—quite unexpected observation, as the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cthermo}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex adopts a closed (rod-shape) in a resting state 
\begin_inset CommandInset citation
LatexCommand citep
key "Rotheneder2023"
literal "true"

\end_inset

, rather than the open conformation that is observed for the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ec}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 complex 
\begin_inset CommandInset citation
LatexCommand citep
key "Kashammer2019"
literal "true"

\end_inset

.
 The open conformation of the complex was believed to be the conformation
 that is loaded onto 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

, however,  at least in the case of the eukaryotic 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 the `rod` conformation is proposed as a loading conformation.
 
\begin_inset CommandInset citation
LatexCommand cite
key "Rotheneder2023"
literal "true"

\end_inset

 propose that the C-terminal fragment of eukaryotic 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 is responsible for the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

–independent 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 binding.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 C terminus could bind 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 directly, or orchestrate the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

–binding of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 catalytic core 
\begin_inset CommandInset citation
LatexCommand citep
key "Williams2008"
literal "true"

\end_inset

.
 However, the presented data show that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 has to be passed through the coiled coils of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 to the catalytic core of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

.
 This suggests that at least two different 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

–binding models must exist—an observation that fits well with the single-molecul
e 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 curtain experiments—
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 binds 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 ends in an 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-dependent fashion, cleavage of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ku}
\end_layout

\end_inset

–blocked end (and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 itself) is an 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-dependent process as well, but the facilitated one-dimensional diffusion
 over the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 is 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-independent 
\begin_inset CommandInset citation
LatexCommand citep
key "Myler2017"
literal "true"

\end_inset

.
 Moreover, the C terminus of human 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 was found to be cleaved by the DNA-dependent metalloprotease SPRTN in cancer
 cells—
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 lacking its C terminus is suggested to be a reason for impaired 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 binding and increased radiosensitivity 
\begin_inset CommandInset citation
LatexCommand citep
key "Na2021"
literal "true"

\end_inset

.
 
\end_layout

\begin_layout Section
Coiled coil of the Rad50—flexible linker between the zinc hook and catalytic
 core
\end_layout

\begin_layout Standard
Another unique part of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex is the extremely long coiled coil region of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 protein.
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
MarginFig{gfx/mrn_dimer.png}{
\backslash
Gls{mrn}--
\backslash
gls{mrn} dimer (based on 
\backslash
href{https://www.rcsb.org/structure/7ZQY}{7ZQY}).}{fig:gfx/mrn_dimer.png}
\end_layout

\end_inset

The long internal coiled coil that constitutes the middle part of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 is formed by folding upon itself in the middle of the protein (the zinc
 hook), so the antiparallel coiled coil is formed in an intermolecular fashion.
 Atomic force microscopy showed that the coiled coil domains of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ec}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 span around 400
\begin_inset space \space{}
\end_inset


\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
AA
\end_layout

\end_inset

, while the coiled coil domain from human 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 is around 100
\begin_inset space \space{}
\end_inset


\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
AA
\backslash

\end_layout

\end_inset

 longer and reaches up to 500
\begin_inset space \space{}
\end_inset


\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
AA
\end_layout

\end_inset

.
 The length and the sequence of coiled coils are quite well conserved, moreover,
 the truncation of the coiled coils leads to severe biological consequences—shor
tening of the coiled coil of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 abrogates the physiological meiotic 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dsb}
\end_layout

\end_inset

 formation, telomere maintenance, and impairs the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 repair by 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nhej}
\end_layout

\end_inset

, interestingly 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hr}
\end_layout

\end_inset

 abrogation appeared after the deletion of the longer fragment than the
 deleted fragment that caused the problems with 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nhej}
\end_layout

\end_inset

 
\begin_inset CommandInset citation
LatexCommand citep
key "Hohl2010"
literal "true"

\end_inset

.
\end_layout

\begin_layout Standard
The atomic force microscopy and the recent 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cryoem}
\end_layout

\end_inset

 images present the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complexes in various conformations.
 Those conformations are available to form due to the flexibility of the
 coiled coil, this is probably achieved by breaks in heptade repeats of
 the coiled coil.
 The coiled coil conformation is dependent on the binding of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 in the globular domain of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 or 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

.
 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Kashammer2019"
literal "true"

\end_inset

 have observed that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ec}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 in the absence of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 the nuclease domains were separated, and the stable globular domain formation
 was obtained after introduction of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 to the solution.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-bound complex is proposed as a major autoinhibited state of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 complex (
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 active sites are blocked), due to the abundance of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 in the cell and the low efficiency of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 in 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-hydrolysis.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-bound 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 presents a more compact structure, however,  the coiled coils of each 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 protomer are separated, and form the flexible (thus so far impossible to
 use as input in the model reconstruction) `open` conformation.
 Further complex stiffening was observed in the vitrified sample containing
 both 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

— the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 complex was observed in the rod-shaped conformation, which allowed to reconstru
ct around one-third (from globular domains to the zinc hook) of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

 structure.
\end_layout

\begin_layout Standard
\begin_inset Note Note
status open

\begin_layout Plain Layout
https://data.mendeley.com/datasets/v93tby2spy/1/files/5272560d-7ef0-4cd3-8128-99b9
86485581 micrograph
\end_layout

\end_inset


\end_layout

\begin_layout Standard
However,  as has been mentioned previously, the eukaryotic 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 behaves in a different manner than the prokaryotic 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mr}
\end_layout

\end_inset

, the resting, 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{atp}
\end_layout

\end_inset

-bound state of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 assumes mostly a rod-shape (however,  the open coiled coil subpopulation
 is observed as well).
 
\begin_inset CommandInset citation
LatexCommand citep
key "Rotheneder2023"
literal "true"

\end_inset

 
\end_layout

\begin_layout Section
The zinc hook—a tetramerization domain?
\begin_inset CommandInset label
LatexCommand label
name "sec:The-zinc-hook"

\end_inset


\end_layout

\begin_layout Standard
The atomic force microscopy of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 showed that at the apex of the coiled coil domain, the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 interacts with another 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 protomer, the follow-up X-ray crystallography studies (and more recent
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cryoem}
\end_layout

\end_inset

) showed the detailed structure of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{ppi}
\end_layout

\end_inset

—the zinc hook of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

.
 The 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{interface}
\end_layout

\end_inset

 formed by two 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 protomers is a 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{zppi}
\end_layout

\end_inset

, where two of the protomers co-coordinate a single Zn(II) ion
\begin_inset Foot
status collapsed

\begin_layout Plain Layout
The first structure of the zinc hook presents zinc hook from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 (
\begin_inset CommandInset href
LatexCommand href
name "1L8D"
target "https://www.rcsb.org/structure/1L8D"
literal "false"

\end_inset

), the structure contains Hg(II) ion, instead of Zn(II), this is discussed
 more in 
\begin_inset Flex CT - auto cross-reference
status open

\begin_layout Plain Layout
\begin_inset CommandInset ref
LatexCommand labelonly
reference "chap:HgRad50"
plural "false"
caps "false"
noprefix "false"

\end_inset


\end_layout

\end_inset

.
\end_layout

\end_inset

, by the conserved CXXC motif, where C corresponds to a cysteine residue,
 and X corresponds to any of 20 canonical amino acids.
 The Zn(II) co-coordination of the cysteine's thiolates implies that Zn(II)
 directly takes part in the formation of the tertiary structure of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, what is more, the Zn(II) ion induces metal–coupled folding of the zinc
 hook domain, which is one of the reasons of the extreme stability of the
 Zn(
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

)
\begin_inset script subscript

\begin_layout Plain Layout
2
\end_layout

\end_inset

 complex.
 
\begin_inset CommandInset citation
LatexCommand citep
key "Kochanczyk2016,Kochanczyk2016a"
literal "true"

\end_inset


\end_layout

\begin_layout Standard
The zinc hook domain is not structurally separated from the coiled coil
 domain of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, i.e., one cannot separate the particular domain just by assessment of the
 structure like in the case of pyruvate kinase (
\begin_inset CommandInset href
LatexCommand href
name "1PKN"
target "https://www.rcsb.org/structure/1PKN"
literal "false"

\end_inset

), where three domain structure is easily seen at once 
\begin_inset CommandInset citation
LatexCommand citep
key "Morris2022"
literal "true"

\end_inset

.
 A similar distinction between the globular domain of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 and the superhelical domain is visible without any further investigation
 (see 
\begin_inset CommandInset href
LatexCommand href
name "7ZR1"
target "https://www.rcsb.org/structure/7ZR1"
literal "false"

\end_inset

).
 However,  the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{UniProt}
\end_layout

\end_inset

 marks 
\emph on
a priori
\emph default
 the stretch of around 100 amino acids flanking the canonical CXXC motif
 to be the zinc hook, however,  this approach seems to be experimentally
 unjustified.
\end_layout

\begin_layout Standard
In structural biology, protein domain is understood as a part of the protein
 that is folded independently of the rest of the protein, and is stable
 
\emph on
per se
\emph default
.
 Combining this with the 
\emph on
a priori
\emph default
 assignment of the zinc hook domain is not justified in the lack of the
 structural discriminant of the coiled coil and the zinc hook domain.
 The marking of the region that makes the zinc hook domain should be 
\emph on
functional 
\emph default
(e.g.
 assessment of the stability), not structural.
 This distinction is possible due to the experimental work performed by
 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Kochanczyk2016"
literal "true"

\end_inset

 and complemented further by 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Padjasek2020a"
literal "true"

\end_inset

 for the zinc hook domain from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

.
 
\begin_inset CommandInset citation
LatexCommand citep
key "Kochanczyk2016,Kochanczyk2016a,Padjasek2020a"
literal "true"

\end_inset

 The stabilities studies show that in the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

 only a 45-amino-acid-long fragment (with the CXXC motif and flanking region)
 is sufficient to form a stable domain, further elongation does not result
 in the stabilization, thus the zinc hook in the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{UniProt}
\end_layout

\end_inset

 should be annotated as this short region in case of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

.
 The stabilities studies performed during my PhD project show that in the
 case of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 the zinc hook domain is longer—an increment in the stability of the zinc
 hook domain is observed up to a length of ~130 amino acids (see 
\begin_inset Flex CT - auto cross-reference
status open

\begin_layout Plain Layout
\begin_inset CommandInset ref
LatexCommand labelonly
reference "chap:hRad50"
plural "false"
caps "false"
noprefix "false"

\end_inset


\end_layout

\end_inset

).
 
\begin_inset CommandInset citation
LatexCommand citep
key "Tran2022"
literal "true"

\end_inset

 
\end_layout

\begin_layout Standard
From  the first experiments investigating the zinc hook from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, till the beginning of 
\begin_inset CommandInset citation
LatexCommand citebyear
key "Rotheneder2023"
literal "true"

\end_inset

 the zinc hook was thought to be a dimerization domain—Zn(II) is bound in
 a tetrahedron geometry, by two cysteines from each 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 protomer.
 The 
\begin_inset CommandInset href
LatexCommand href
name "1L8D"
target "https://www.rcsb.org/structure/1L8D"
literal "false"

\end_inset

 
\begin_inset CommandInset citation
LatexCommand citep
key "Hopfner2002"
literal "true"

\end_inset

 structure presented by Karl-Peter Hopfner after his postdoctoral fellowship
 presents the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

 in a `V-shaped` conformation, without any possibilities of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 tetramerization.
 After 15 years, 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Park2017"
literal "true"

\end_inset

 present the structure of a zinc hook from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

—
\begin_inset CommandInset href
LatexCommand href
name "5GOX"
target "https://www.rcsb.org/structure/5GOX"
literal "false"

\end_inset

, the structure adopts a totally different shape, compared to the V-shaped
 conformation of the zinc hook from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

.
 The newly described `rod` conformation is validated by the in-solution
 experiments (
\emph on
inter alia
\emph default
 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{saxs}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{FRET}
\end_layout

\end_inset

) and stands in agreement with the atomic force microscopy of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

.
 
\begin_inset CommandInset citation
LatexCommand citep
key "Park2017"
literal "true"

\end_inset

 Four years later 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Soh2021"
literal "true"

\end_inset

 present an almost identical sequence of 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 to the sequence crystallized in the 
\begin_inset CommandInset href
LatexCommand href
name "1L8D"
target "https://www.rcsb.org/structure/1L8D"
literal "false"

\end_inset

 (
\begin_inset CommandInset href
LatexCommand href
name "6ZFF"
target "https://www.rcsb.org/structure/6ZFF"
literal "false"

\end_inset

), however,  the zinc hook, instead of adopting the `V-shaped` assembly,
 adopts a `rod-shape`.
 
\begin_inset CommandInset citation
LatexCommand citep
key "Soh2021"
literal "true"

\end_inset

 The 
\begin_inset CommandInset href
LatexCommand href
name "6ZFF"
target "https://www.rcsb.org/structure/6ZFF"
literal "false"

\end_inset

 structure has suggested that the zinc hook domain from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{pf}
\end_layout

\end_inset

 has stabilizing interactions that are not present in the 
\begin_inset CommandInset href
LatexCommand href
name "1L8D"
target "https://www.rcsb.org/structure/1L8D"
literal "false"

\end_inset

, however,  this does not stand in agreement with the experimental data
 provided by 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Kochanczyk2016"
literal "true"

\end_inset

 and 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Padjasek2020a"
literal "true"

\end_inset

,
\begin_inset CommandInset citation
LatexCommand citep
key "Kochanczyk2016,Kochanczyk2016a,Padjasek2020a"
literal "true"

\end_inset

 this is discussed in the 
\begin_inset CommandInset citation
LatexCommand citetitle
key "Tran2022"
literal "true"

\end_inset

 (see 
\begin_inset Flex CT - auto cross-reference
status open

\begin_layout Plain Layout
\begin_inset CommandInset ref
LatexCommand labelonly
reference "chap:hRad50"
plural "false"
caps "false"
noprefix "false"

\end_inset


\end_layout

\end_inset

), as the experimental description of the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 zinc hook stability stands in a great agreement with the 
\begin_inset CommandInset href
LatexCommand href
name "5GOX"
target "https://www.rcsb.org/structure/5GOX"
literal "false"

\end_inset

 structure proposed by 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Park2017"
literal "true"

\end_inset

, making a great pivot for discussion of the relationship between the structure
 and sequence.
 After 21 years, Hopfner, now as a group leader, releases another 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 structure.
 Those 21 years of technological development allowed for the reconstruction
 of the 
\emph on
complete
\emph default
 eukaryotic 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 structure, together in complex with 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mre}
\end_layout

\end_inset

 and 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{nbs}
\end_layout

\end_inset

 (
\begin_inset CommandInset href
LatexCommand href
name "7ZR1"
target "https://www.rcsb.org/structure/7ZR1"
literal "false"

\end_inset

).
 The observed 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cthermo}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex, similar to 
\begin_inset CommandInset href
LatexCommand href
name "5GOX"
target "https://www.rcsb.org/structure/5GOX"
literal "false"

\end_inset

 and 
\begin_inset CommandInset href
LatexCommand href
name "6ZFF"
target "https://www.rcsb.org/structure/6ZFF"
literal "false"

\end_inset

 presents the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 in the `rod` conformation.
 While taking 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cryoem}
\end_layout

\end_inset

 microphotographs 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Rotheneder2023"
literal "true"

\end_inset

 observed that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex formed conglomerates, connected by the zinc hook, however,  due
 to the low number of particles it was impossible to resolve the three-dimension
al map of the conglomerate, to obtain a structure with better resolution
 and to investigate the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{interface}
\end_layout

\end_inset

 between the apexes, the crystal structure of the zinc hook from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cthermo}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 was resolved (
\begin_inset CommandInset href
LatexCommand href
name "7ZQY"
target "https://www.rcsb.org/structure/7ZQY"
literal "false"

\end_inset

).
 It turned out that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{asunit}
\end_layout

\end_inset

 consist of the dimer of dimers (zinc hooks)—the same arrangement that was
 observed by the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{cryoem}
\end_layout

\end_inset

.
 Curiously enough the previous eukaryotic zinc hook structure, 
\begin_inset CommandInset href
LatexCommand href
name "5GOX"
target "https://www.rcsb.org/structure/5GOX"
literal "false"

\end_inset

, shows the same tendency in the crystal lattice (when the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{asunit}
\end_layout

\end_inset

 is duplicated and translated), however,  the propensity to form tetrameric
 assembly was not obvious to 
\begin_inset CommandInset citation
LatexCommand citeauthor
key "Park2017"
literal "true"

\end_inset

 when the structure was published, thus the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{bassembly}
\end_layout

\end_inset

 presented in the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rcsb}
\end_layout

\end_inset

 shows the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

 in a dimeric assembly.
 During the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{saxs}
\end_layout

\end_inset

 experiments performed by me on the zinc hook from 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hs}
\end_layout

\end_inset

 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{rad}
\end_layout

\end_inset

, I was not able to observe the tetramerization, as the radius of gyration
 stands in accordance with the crystal structure.
 
\begin_inset CommandInset citation
LatexCommand citep
key "Tran2022"
literal "true"

\end_inset

 
\end_layout

\begin_layout Standard
The ability to form various architectures by interaction in the zinc hook
 domain suggests that the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{mrn}
\end_layout

\end_inset

 complex is able to assume conformations that could hold the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 fragments together, which possible could be one of the steps involved in
 the 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{dna}
\end_layout

\end_inset

 damage repair via 
\begin_inset ERT
status open

\begin_layout Plain Layout


\backslash
gls{hr}
\end_layout

\end_inset

.
 The architecture formed by the tetramerization of the 
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 by apex-apex interaction could form a structure that is at least 1000
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AA
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 long, which theoretically should allow the binding of the sister chromatids
 during the 
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.
 However,  to date the exact biological role of the apex-apex tetramerization
 of the 
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 is shrouded in mystery—the apex-apex interface is not conserved evolutionarily,
 and the tethering and its regulation probably differ in organisms.
 Precise 
\emph on
in vivo 
\emph default
experiments are needed to put the 
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 tetramerization in the biological aspect.
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\end_document