共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
3.
4.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
5.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
6.
7.
As obligate intracellular parasites, viruses exploit diverse cellular
signaling machineries, including the mitogen-activated protein-kinase pathway,
during their infections. We have demonstrated previously that the open reading
frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90
ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities
(Kuang, E., Tang, Q., Maul, G. G., and Zhu, F.
(2008) J. Virol. 82
,1838
-1850). Here, we define the
mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45
to RSK increases the association of extracellular signal-regulated kinase
(ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass
protein complexes. We further demonstrated that the complexes shielded active
pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK
and ERK were activated and sustained at high levels. Finally, we provide
evidence that this mechanism contributes to the sustained activation of ERK
and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase
(ERK)2
mitogen-activated protein kinase (MAPK) signaling pathway has been implicated
in diverse cellular physiological processes including proliferation, survival,
growth, differentiation, and motility
(1-4)
and is also exploited by a variety of viruses such as Kaposi
sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human
immunodeficiency virus, respiratory syncytial virus, hepatitis B virus,
coxsackie, vaccinia, coronavirus, and influenza virus
(5-17).
The MAPK kinases relay the extracellular signaling through sequential
phosphorylation to an array of cytoplasmic and nuclear substrates to elicit
specific responses (1,
2,
18). Phosphorylation of MAPK
is reversible. The kinetics of deactivation or duration of signaling dictates
diverse biological outcomes
(19,
20). For example, sustained
but not transient activation of ERK signaling induces the differentiation of
PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells
(20-22).
During viral infection, a unique biphasic ERK activation has been observed for
some viruses (an early transient activation triggered by viral binding or
entry and a late sustained activation correlated with viral gene expression),
but the responsible viral factors and underlying mechanism for the sustained
ERK activation remain largely unknown
(5,
8,
13,
23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine
kinases that lie at the terminus of the ERK pathway
(1,
24-26).
In mammals, four isoforms are known, RSK1 to RSK4. Each one has two
catalytically functional kinase domains, the N-terminal kinase domain (NTKD)
and C-terminal kinase domain (CTKD) as well as a linker region between the
two. The NTKD is responsible for phosphorylation of exogenous substrates, and
the CTKD and linker region regulate RSK activation
(1,
24,
25). In quiescent cells ERK
binds to the docking site in the C terminus of RSK
(27-29).
Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase
(MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker
region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD
activation loop. The activated CTKD then phosphorylates Ser-380 in the linker
region, creating a docking site for 3-phosphoinositide-dependent protein
kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates
Ser-221 of RSK in the activation loop and activates the NTKD. The activated
NTKD autophosphorylates the serine residue near the ERK docking site, causing
a transient dissociation of active ERK from RSK
(25,
26,
28). The stimulation of
quiescent cells by a mitogen such as epidermal growth factor or a phorbol
ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually
results in a transient RSK activation that lasts less than 30 min. RSKs have
been implicated in regulating cell survival, growth, and proliferation.
Mutation or aberrant expression of RSK has been implicated in several human
diseases including Coffin-Lowry syndrome and prostate and breast cancers
(1,
24,
25,
30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma,
primary effusion lymphoma, and a subset of multicentric Castleman disease
(33,
34). Infection and
reactivation of KSHV activate multiple MAPK pathways
(6,
12,
35). Noticeably, the ERK/RSK
activation is sustained late during KSHV primary infection and reactivation
from latency (5,
6,
12,
23), but the mechanism of the
sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45,
an immediate early and also virion tegument protein of KSHV, interacts with
RSK1 and RSK2 and strongly stimulates their kinase activities
(23). We also demonstrated
that the activation of RSK plays an essential role in KSHV lytic replication
(23). In the present study we
determined the mechanism of ORF45-induced sustained ERK/RSK activation. We
found that ORF45 increases the association of RSK with ERK and protects them
from dephosphorylation, causing sustained activation of both ERK and RSK. 相似文献
8.
Nodar Makharashvili Tian Mi Olga Koroleva Sergey Korolev 《The Journal of biological chemistry》2009,284(3):1425-1434
RecF pathway proteins play an important role in the restart of stalled
replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR,
and RecO initiate homologous recombination (HR) by loading of the RecA
recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein.
The specific role of RecF in this process is not well understood. Previous
studies have proposed that RecF directs the RecOR complex to boundaries of
damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA
junctions. RecF belongs to ABC-type ATPases, which function through an
ATP-dependent dimerization. Here, we demonstrate that the RecF of
Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer,
and that the DNA binding and ATPase activity of RecF depend on both the
structure of DNA substrate, and the presence of RecR. We found that RecR
interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity
to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to
ssDNA and dimerization, likely due to increasing the ATPase rate. The
DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific
protein-protein interactions without significant contributions from RecR-DNA
interactions. Finally, RecF neither alone nor in complex with RecR
preferentially binds to the ss/dsDNA junction. Our data suggest that the
specificity of the RecFOR complex toward the boundaries of DNA damaged regions
may result from a network of protein-protein and DNA-protein interactions,
rather than a simple recognition of the ss/dsDNA junction by RecF.Homologous recombination
(HR)2 is one of the
primary mechanisms by which cells repair dsDNA breaks (DSBs) and ssDNA gaps
(SSGs), and is important for restart of stalled DNA replication
(1). HR is initiated when
RecA-like recombinases bind to ssDNA forming an extended nucleoprotein
filament, referred to as a presynaptic complex
(2). The potential for genetic
rearrangements dictates that HR initiation is tightly regulated at multiple
levels (1). During replication,
the ssDNA-binding protein (SSB) protects transiently unwound DNA chains,
preventing interactions with recombinases. Following DNA damage, recombination
mediator proteins (RMPs) initiate HR by facilitating the formation of the
recombinase filaments with ssDNA, while removing SSB
(3,
4). Mutations in human proteins
involved in HR initiation are linked to cancer predisposition, chromosome
instability, UV sensitivity, and premature aging diseases
(4–8).
To date, little is known about the mechanism by which RMPs regulate the
formation of the recombinase filaments on the SSB-protected ssDNA.In Escherichia coli, there are two major recombination pathways,
RecBCD and RecF (9,
10). A helicase/nuclease
RecBCD complex processes DSBs and recruits RecA on ssDNA in a
sequence-specific manner
(11–13).
The principle players in the RecF pathway are the RecF, RecO, and RecR
proteins, which form an epistatic group that is important for SSG repair, for
restart of stalled DNA replication, and under specific conditions, can also
process DSBs
(14–20).
Homologs of RecF, -O, and -R are present in the majority of known bacteria
(21), including
Deinococcus radiodurans, extremely radiation-resistant bacteria that
lacks the RecBCD pathway, yet is capable of repairing thousands of DSBs
(22,
23). In addition, the sequence
or functional homologs of RecF pathway proteins are involved in similar
pathways in eukaryotes that include among others WRN, BLM, RAD52, and BRCA2
proteins
(4–8).The involvement of all three RecF, -O, and -R proteins in HR initiation is
well documented by genetic and cellular approaches
(18,
24–30),
yet their biochemical functions in the initiation process remain unclear,
particularly with respect to RecF. RecO and RecR proteins are sufficient to
promote formation of the RecA filament on SSB-bound ssDNA in vitro
(27). The UV-sensitive
phenotype of recF mutants can be suppressed by RecOR overexpression,
suggesting that RecF may direct the RMP complex to DNA-damaged regions where
HR initiation is required
(31). In agreement with this
hypothesis, RecF dramatically increases the efficiency of the RecA loading at
ds/ssDNA junctions with a 3′ ssDNA extension under specific conditions
(32). RecF and RecR proteins
also prevent the RecA filaments from extending into dsDNA regions adjacent to
SSGs (33). These data suggest
that RecF may directly recognize an ss/dsDNA junction structure
(34). However, DNA binding
experiments have not provided clear evidence to support such a hypothesis
(11).The targeting promoted by RecF may also occur through more complex
processes. RecF shares a high structural similarity with the head domain of
Rad50, an ABC-type ATPase that recognizes DSBs and initiates repair in archaea
and eukaryotes (35). All known
ABC-type ATPases function as oligomeric complexes in which a sequence of
inter- and intra-molecular interactions is triggered by the ATP-dependent
dimerization and the dimer-dependent ATP hydrolysis
(36–39).
RecF is also an ATP-dependent DNA-binding protein and a weak DNA-dependent
ATPase (11,
40). RecF forms an
ATP-dependent dimer and all three conserved motifs (Walker A, Walker B, and
“signature”) of RecF are important for ATP-dependent dimerization,
ATP hydrolysis, and functional resistance to DNA damage
(35). Thus, RecF may function
in recombination initiation through a complex pathway of protein-protein and
DNA-protein interactions regulated by ATP-dependent RecF dimerization.In this report, we present a detailed characterization of the RecF
dimerization, and its role in the RecF interaction with various DNA
substrates, with RecR, and in ATP hydrolysis. Our data outline the following
key findings. First, RecF interacts with DNA as a dimer. Second, neither RecF
alone nor the RecFR complex preferentially binds the ss/dsDNA junction.
Finally, RecR changes the ATPase activity and the DNA binding of RecF by
destabilizing the interaction with ssDNA, and greatly enhancing the
interaction with dsDNA. Our results suggest that the specificity of RecF for
the boundaries of SSGs is likely to result from a sequence of protein-protein
interaction events rather than a simple RecF ss/dsDNA binding, underlining a
highly regulated mechanism of the HR initiation by the RecFOR proteins. 相似文献
9.
10.
11.
12.
13.
Motoki Takaku Shinichi Machida Noriko Hosoya Shugo Nakayama Yoshimasa Takizawa Isao Sakane Takehiko Shibata Kiyoshi Miyagawa Hitoshi Kurumizaka 《The Journal of biological chemistry》2009,284(21):14326-14336
The RAD51 protein is a central player in homologous recombinational repair.
The RAD51B protein is one of five RAD51 paralogs that function in the
homologous recombinational repair pathway in higher eukaryotes. In the present
study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested
to be involved in actin-remodeling processes, unexpectedly binds to the RAD51
and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and
strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged
DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone
promotes single-stranded DNA annealing, and the recombination activities of
the EVL protein are further enhanced by the RAD51B protein. The expression of
the EVL protein is not ubiquitous, but it is significantly expressed in breast
cancer-derived MCF7 cells. These results suggest that the EVL protein is a
novel recombination factor that may be required for repairing specific DNA
lesions, and that may cause tumor malignancy by its inappropriate
expression.Chromosomal DNA double strand breaks
(DSBs)2 are potential
inducers of chromosomal aberrations and tumorigenesis, and they are accurately
repaired by the homologous recombinational repair (HRR) pathway, without base
substitutions, deletions, and insertions
(1–3).
In the HRR pathway (4,
5), single-stranded DNA (ssDNA)
tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue
of the bacterial RecA protein, binds to the ssDNA tail and forms a helical
nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact
double-stranded DNA (dsDNA) to form a three-component complex, containing
ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the
RAD51 protein promotes recombination reactions, such as homologous pairing and
strand exchange
(6–9).The RAD51 protein requires auxiliary proteins to promote the homologous
pairing and strand exchange reactions efficiently in cells
(10–12).
In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the
RAD51 protein
(13–17)
and stimulate the RAD51-mediated homologous pairing and/or strand exchange
reactions in vitro
(18–21).
The human RAD51AP1 protein, which directly binds to the RAD51 protein
(22), was also found to
stimulate RAD51-mediated homologous pairing in vitro
(23,
24). The BRCA2 protein
contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs
(25–33),
and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated
RAD51-mediated strand exchange
(34,
35). Most of these
RAD51-interacting factors are known to be required for efficient RAD51
assembly onto DSB sites in cells treated with ionizing radiation
(10–12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which
share about 20–30% amino acid sequence similarity with the RAD51 protein
(36–38).
RAD51B-deficient cells are hypersensitive to DSB-inducing agents,
such as cisplatin, mitomycin C (MMC), and γ-rays, indicating that the
RAD51B protein is involved in the HRR pathway
(39–44).
Genetic experiments revealed that RAD51B-deficient cells exhibited
impaired RAD51 assembly onto DSB sites
(39,
44), suggesting that the
RAD51B protein functions in the early stage of the HRR pathway. Biochemical
experiments also suggested that the RAD51B protein participates in the early
to late stages of the HRR pathway
(45–47).In the present study, we found that the human EVL (Ena/Vasp-like) protein
binds to the RAD51 and RAD51B proteins in a HeLa cell extract. The EVL protein
is known to be involved in cytoplasmic actin remodeling
(48) and is also overexpressed
in breast cancer (49). Like
the RAD51B knockdown cells, the EVL knockdown cells partially impaired RAD51
foci formation after DSB induction, suggesting that the EVL protein enhances
RAD51 assembly onto DSB sites. The purified EVL protein preferentially bound
to ssDNA and stimulated RAD51-mediated homologous pairing and strand exchange.
The EVL protein also promoted the annealing of complementary strands. These
recombination reactions that were stimulated or promoted by the EVL protein
were further enhanced by the RAD51B protein. These results strongly suggested
that the EVL protein is a novel factor that activates RAD51-mediated
recombination reactions, probably with the RAD51B protein. We anticipate that,
in addition to its involvement in cytoplasmic actin dynamics, the EVL protein
may be required in homologous recombination for repairing specific DNA
lesions, and it may cause tumor malignancy by inappropriate recombination
enhanced by EVL overexpression in certain types of tumor cells. 相似文献
14.
15.
16.
Rebecca A. Chanoux Bu Yin Karen A. Urtishak Amma Asare Craig H. Bassing Eric J. Brown 《The Journal of biological chemistry》2009,284(9):5994-6003
Chromosomal abnormalities are frequently caused by problems encountered
during DNA replication. Although the ATR-Chk1 pathway has previously been
implicated in preventing the collapse of stalled replication forks into
double-strand breaks (DSB), the importance of the response to fork collapse in
ATR-deficient cells has not been well characterized. Herein, we demonstrate
that, upon stalled replication, ATR deficiency leads to the phosphorylation of
H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of
homologous recombination and fork restart. Because H2AX has been shown to play
a facilitative role in homologous recombination, we hypothesized that H2AX
participates in Rad51-mediated suppression of DSBs generated in the absence of
ATR. Consistent with this model, increased Rad51 focal accumulation in
ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR
and H2AX lead to synergistic increases in chromatid breaks and translocations.
Importantly, the ATM and DNA-PK phosphorylation site on H2AX
(Ser139) is required for genome stabilization in the absence of
ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal
role in suppressing DSBs during DNA synthesis in instances of ATR pathway
failure. These results imply that ATR-dependent fork stabilization and
H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress
double-strand breaks as a layered response network when replication
stalls.Genome maintenance prevents mutations that lead to cancer and age-related
diseases. A major challenge in preserving genome integrity occurs in the
simple act of DNA replication, in which failures at numerous levels can occur.
Besides the mis-incorporation of nucleotides, it is during this phase of the
cell cycle that the relatively stable double-stranded nature of DNA is
temporarily suspended at the replication fork, a structure that is susceptible
to collapse into
DSBs.2 Replication
fork stability is maintained by a variety of mechanisms, including activation
of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of
extended stretches of single-stranded DNA at stalled replication forks
(1-4).
Genome maintenance functions for ATR and orthologs in yeast were first
indicated by increased chromatid breaks in ATR-/- cultured cells
(5) and by the
“cut” phenotype observed in Mec1 (Saccharomyces
cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants
(6-9).
Importantly, subsequent studies in S. cerevisiae demonstrated that
mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased
chromosome breaks at regions of the genome that are inherently difficult to
replicate (10), and a
decreased ability to reinitiate replication fork progression following DNA
damage or deoxyribonucleotide depletion
(11-14).In vertebrates, similar replication fork stabilizing functions have been
demonstrated for ATR and the downstream protein kinase Chk1
(15-20).
Several possible mechanisms have been put forward to explain how ATR-Chk1 and
orthologous pathways in yeast maintain replication fork stability, including
maintenance of replicative polymerases (α, δ, and ε) at forks
(17,
21), regulation of branch
migrating helicases, such as Blm
(22-25),
and regulation of homologous recombination, either positively or negatively
(26-29).Consistent with the role of the ATR-dependent checkpoint in replication
fork stability, common fragile sites, located in late-replicating regions of
the genome, are significantly more unstable (5-10-fold) in the absence of ATR
or Chk1 (19,
20). Because these sites are
favored regions of instability in oncogene-transformed cells and preneoplastic
lesions (30,
31), it is possible that the
increased tumor incidence observed in ATR haploinsufficient mice
(5,
32) may be related to subtle
increases in genomic instability. Together, these studies indicate that
maintenance of replication fork stability may contribute to tumor
suppression.It is important to note that prevention of fork collapse represents an
early response to problems occurring during DNA replication. In the event of
fork collapse into DSBs, homologous recombination (HR) has also been
demonstrated to play a key role in genome stability during S phase by
catalyzing recombination between sister chromatids as a means to re-establish
replication forks (33).
Importantly, a facilitator of homologous recombination, H2AX, has been shown
to be phosphorylated under conditions that cause replication fork collapse
(18,
34).Phosphorylation of H2AX occurs predominantly upon DSB formation
(34-38)
and has been reported to require ATM, DNA-PKcs, or ATR, depending on the
context
(37-42).
Although H2AX is not essential for HR, studies have demonstrated that H2AX
mutation leads to deficiencies in HR
(43,
44), and suppresses events
associated with homologous recombination, such as the focal accumulation of
Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin
ubiquitination (43,
45-51).
Therefore, through its contribution to HR, it is possible that H2AX plays an
important role in replication fork stability as part of a salvage pathway to
reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and
H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX
would be expected to dramatically enhance the accumulation of DSBs upon
replication fork stalling. Herein, we utilize both partial and complete
elimination of ATR and H2AX to demonstrate that these genes work cooperatively
in non-redundant pathways to suppress DSBs during S phase. As discussed, these
studies imply that the various components of replication fork protection and
regeneration cooperate to maintain replication fork stability. Given the large
number of genes involved in each of these processes, it is possible that
combined deficiencies in these pathways may be relatively frequent in humans
and may synergistically influence the onset of age-related diseases and
cancer. 相似文献
17.
18.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
19.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
20.
Susan R. Ferrari Jennifer Grubb Douglas K. Bishop 《The Journal of biological chemistry》2009,284(18):11766-11770
During homologous recombination, a number of proteins cooperate to catalyze
the loading of recombinases onto single-stranded DNA. Single-stranded
DNA-binding proteins stimulate recombination by coating single-stranded DNA
and keeping it free of secondary structure; however, in order for recombinases
to load on single-stranded-DNA-binding protein-coated DNA, the activity of a
class of proteins known as recombination mediators is required. Mediator
proteins coordinate the handoff of single-stranded DNA from single-stranded
DNA-binding protein to recombinase. Here we show that a complex of Mei5 and
Sae3 from Saccharomyces cerevisiae preferentially binds
single-stranded DNA and relieves the inhibition of the strand assimilation and
DNA binding abilities of the meiotic recombinase Dmc1 imposed by the
single-stranded DNA-binding protein replication protein A. Additionally, we
demonstrate the physical interaction of Mei5-Sae3 with replication protein A.
Our results, together with previous in vivo studies, indicate that
Mei5-Sae3 is a mediator of Dmc1 assembly during meiotic recombination in
S. cerevisiae.During meiosis, recombination between homologous chromosomes ensures proper
segregation into haploid products. Recombination events are initiated by the
formation of double strand breaks
(DSBs)2 in DNA
(1). This is followed by
resection of free DNA ends to yield 3′ single-stranded tails, upon which
recombinase assembles to form nucleoprotein filaments. Following recombinase
assembly, the nucleoprotein filament engages a donor chromatid, searches for
homologous DNA sequences on that chromatid, and promotes strand exchange to
yield a heteroduplex DNA intermediate often referred to as a joint molecule.
Although recombinase alone is capable of promoting homology search and strand
exchange in vitro, genetic and biochemical studies have demonstrated
that normal recombinase function in vivo requires the activity of a
number of accessory factors
(2). These factors enhance the
assembly of nucleoprotein filaments, target capture, homology search, and
dissociation of recombinase from duplex DNA.Most eukaryotes possess two recombinases, both homologues of the
Escherichia coli recombinase RecA: Rad51, which is the major
recombinase in mitotic cells and is also important during meiotic
recombination, and Dmc1, which functions only in meiosis. Dmc1 and Rad51 have
been shown to assemble at DSBs by immunofluorescence and chromatin
immunoprecipitation
(3–6),
and both proteins oligomerize on single-stranded DNA (ssDNA) to form
nucleofilaments that catalyze strand invasion
(7–9).A number of biochemical studies have defined the role of accessory factors
in stimulating the activity of Rad51
(10–12).
Replication protein A (RPA), the yeast ssDNA-binding protein (SSB), removes
secondary structure in ssDNA that otherwise prevents formation of fully
functional nucleoprotein filaments
(13). Both Rad52 protein
(11,
12) and the heterodimeric
protein Rad55/Rad57 (14) can
overcome the inhibitory effect of RPA on Rad51 nucleoprotein filament
formation in purified systems, mediating a handoff between RPA and Rad51. It
is thought that the mechanism for the mediator activity of Rad52 involves
Rad52 recognizing and binding to RPA-coated ssDNA, where it provides
nucleation sites for the recruitment of free molecules of Rad51
(15). The tumor suppressor
protein BRCA2 also serves as an assembly factor for Rad51 during mitosis in a
variety of species that encode orthologues of this protein, including mice
(16), corn smut
(17), and humans
(18).The meiosis-specific recombinase Dmc1 is stimulated by a distinct set of
accessory factors. Immunostaining studies suggest that the Rad51 mediators
Rad52 and Rad55/Rad57 are not required for assembly of Dmc1 foci in
vivo, although Rad51 itself promotes Dmc1 foci
(19–21).
More recently, immunostaining and chromatin immunoprecipitation experiments
demonstrated a role for the Mei5 and Sae3 proteins of Saccharomyces
cerevisiae in assembly of Dmc1 at sites of DSBs in vivo
(22,
23). Consistent with these
observations, mei5 and sae3 mutants display markedly similar
meiotic defects as compared with dmc1 mutants, including defects in
sporulation, spore viability, crossing over, DSB repair, progression through
meiosis, and synaptonemal complex formation
(19,
22–24).
Finally, the three proteins have been shown to physically interact; Mei5 and
Sae3 have been co-purified and co-immunoprecipitated, and an N-terminal
portion of Mei5 has been shown to interact with Dmc1 in a two-hybrid assay
(22).The fission yeast Schizosaccharomyces pombe encodes two proteins,
Swi5 and Sfr1, which share sequence homology with Sae3 and Mei5, respectively
(22). Swi5 and Sfr1 have been
shown to stimulate the strand exchange activity of Rhp51 (the S.
pombe Rad51 homologue) and Dmc1
(25). Although some results
indicate functional similarity of Swi5-Sfr1 and Mei5-Sae3, there are also
clear differences. The Mei5-Sae3 complex of budding yeast is expressed solely
during meiosis, and no mitotic phenotypes have been reported for mei5
or sae3 mutants (22,
24,
26). In contrast, the
Swi5-Sfr1 complex of fission yeast is expressed in mitotic and meiotic cells,
and mutations in SWI5 have been shown to cause defects in mitotic
recombination (27).
Furthermore, although mei5 and sae3 mutants are
phenotypically similar to dmc1 mutants, swi5 and
sfr1 mutants display more severe meiotic defects during fission yeast
meiosis than do dmc1 mutants
(27–29).
These data suggest that although Swi5-Sfr1 clearly contributes to Rad51
activity in fission yeast, it is possible that the activity of Mei5-Sae3 is
restricted to stimulating Dmc1 in budding yeast.In this study, a biochemical approach is used to test the budding yeast
Mei5-Sae3 complex for properties expected of a recombinase assembly mediator.
We show that Mei5-Sae3 binds both ssDNA and double-stranded DNA (dsDNA) but
binds ssDNA preferentially. We also show that Mei5-Sae3 can overcome the
inhibitory effects of RPA on the ssDNA binding and strand assimilation
activities of Dmc1. Finally, we show that Mei5-Sae3 and RPA bind one another
directly. These results indicate that Mei5-Sae3 acts directly as a mediator
protein for assembly of Dmc1. 相似文献