Biology Reference
In-Depth Information
Formation and maintenance of a stable nucleoprotein filament (NPF) is
required for the DNA homology search and strand exchange by RecA/RAD51
recombinases.
85,99,100
RAD51 nucleates on both ssDNA and dsDNA
in vitro
,
forming an extended NPF.
101,102
Biochemical studies have shown that incor-
poration of monovalent salts in the reaction buffer biases RAD51 nucleation on
ssDNA.
103,104
This observation appears to presage the possibility that other
recombination mediators might be responsible for efficient nucleation of
RAD51 on ssDNA. A single RAD51 molecule binds to 3-4 nt or bp, extending
the helical pitch by
50% compared to canonical B-form DNA.
101,102
Interest-
ingly, when calcium was substituted for magnesium, human RAD51 displayed
enhanced strand exchange activity
in vitro
.
100
This effect has been attributed to
the suppression of ATP hydrolysis by the calcium, which appears to enhance the
lifetime of the ATP-bound active NPF.
100
This calcium-mediated stimulation is
unique to human RAD51.
100
Budding yeast containing a
rad51
deletion is viable.
86
However, knockout
of RAD51 in vertebrates leads to chromosomal instability and embryonic
lethality.
51,52
Although no mutations have been reported in the open reading
frame, in many cancers and cancer cell lines the expression of RAD51 is
increased, presumably providing a replicative advantage to the rapidly dividing
cells via its role in the HR repair of collapsed forks.
105-107
A catalytically conserved lysine residue in the Walker A box (yeast Rad51
(K191) and human RAD51(K133)) is essential for ATP binding and hydrolysis.
Mutation of this conserved lysine to alanine (Rad51(K191A)) leads to a null
phenotype and is a dominant negative phenotype in diploid cells.
41,108
Expres-
sion of Rad51(K191R) in
rad51
null yeast strains leads to resistance to DSB-
causing agents, suggesting nucleotide binding is sufficient for HR repair
in vivo
.
109
Human RAD51(K133R) binds ATP but is unable to hydrolyze
ATP, similar to the yeast mutation.
110,111
Overexpression of human RAD51
(K133R) in chicken DT40
RAD51
knockout cells confers partial resistance to
IR.
111
RAD51(K133R) forms a stable NPF on DNA and has enhanced recom-
binase activity
in vitro
.
110
However, generated mouse embryonic stem cells
that express RAD51(K133R) display increased sensitivity to DSBs and reduced
efficiency of spontaneous sister chromatid exchanges.
112
ATP binds at the interface region of two adjacent RAD51 monomers within
the NPF.
95,113
The bottom subunit provides the catalytic Walker A (P-loop)
domain while the top subunit shields the nucleotide with an
ATP cap
contain-
ing a conserved proline residue.
113
For the homology search and strand
exchange, ATP binding but not necessarily ATP hydrolysis is required.
99
When an ATP or a nonhydrolysable ATP analog binds at the subunit interface,
the NPF adopts an active extended conformation. Several biochemical studies
have shown that incorporation of monovalent cations such as ammonium and
potassium as well as divalent cations such as calcium extends the NPF further
