Biology Reference
In-Depth Information
IV. Recombination Mediators
A. RPA
RPA is a heterotrimeric (70 kD, 32 kD, 14 kD) SSB that binds to ssDNA
with high affinity (
Table I
; Refs.
135,136
). It was first shown to stimulate strand
exchange
in vitro
with strand exchange protein 1 (SEP1).
137,138
A similar
stimulatory effect was shown later with yeast Rad51.
139
RPA has a dual stim-
ulatory role during RAD51-mediated strand exchange. During the presynaptic
phase, it binds to ssDNA to prevent secondary structure formation that could
potentially lead to inhibitory effects during RAD51 NPF formation.
140
During
the isoenergetic strand exchange phase, RPA ensures unidirectional heterodu-
plex extension by binding to the displaced ssDNA.
141,142
However, during
recombination assays
in vitro
where only RPA and RAD51 are present, if
RPA is added to ssDNA prior to the addition of RAD51, strand exchange is
inhibited due to the nanomolar affinity of RPA for ssDNA. This inhibition can
be overcome by the addition of recombination mediators such as Rad52 or
Rad55/Rad57 in yeast Rad51 recombinase reactions, and by the addition of
BRCA2 or the RAD51 paralog heterodimer RAD51B-RAD51C in human
RAD51 recombinase reactions
117,119,143-147
(R. Amunugama and R. Fishel,
unpublished data).
B. RAD52
Rad52 plays an essential role in HR and SSA and its deletion leads to severe
sensitivity to DSB-causing agents and defects during meiosis in budding yeast
(
Table I
; Refs.
85,86
). Electron microscopic (EM) evidence indicates that both
yeast and human RAD52 form oligomeric ring structures.
148-150
The EM
structure of human RAD52 indicated that the N terminus is responsible for the
formation of a heptameric ring structure and the C terminus then self-
assembles the heptameric rings into a higher ordered structure.
148,149
However,
two independent X-ray crystallographic analyses revealed that the yeast Rad52
N-terminal residues 1-201 (1-209 of human RAD52) formed an undecameric
(11 subunit) ring.
151,152
The overall structure resembles a mushroom top with
positively charged residues lining a groove on the outside of the ring.
151
Even
though no DNA-containing structures of RAD52 have been solved, the dimen-
sions of the groove indicate that it is large enough to bind ssDNA in a
sequence-independent manner that would position the bases away from the
protein surface for possible annealing with complementary bases.
89,151
The ssDNA-binding property of purified yeast Rad52 was first demonstrated
by Rothstein and colleagues in 1996 and found to reside in the N terminus.
153
A similar ssDNA-binding pattern was observed when ssDNA-RAD52
complexes were probed for hydroxyl radical hypersensitivity.
154
