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1.
组蛋白乙酰化/去乙酰化与基因表达调控 总被引:1,自引:0,他引:1
组蛋白是真核生物染色质的主要成分,组蛋白修饰(如甲基化、乙酰化、磷酸化、泛素化等)在真核生物基因表达调控中发挥着重要的作用.在这些修饰中,组蛋白乙酰化/去乙酰化尤为重要.组蛋白乙酰化/去乙酰化可通过改变染色质周围电荷或参与染色质构型重建而影响基因表达;更重要的是组蛋白乙酰化/去乙酰化可形成一种特殊的“密码”,被其它蛋白质识别,影响多种蛋白质因子的活动或与其相互作用,参与到基因表达调控的整个网络中. 相似文献
2.
Cdt1 Interactions in the Licensing Process: A Model for Dynamic Spatio-temporal Control of Licensing
《Cell cycle (Georgetown, Tex.)》2013,12(13):1549-1552
Within each cell cycle, a cell must ensure that the processes of selection of replication origins (licensing) and initiation of DNA replication are well coordinated to prevent re-initiation of DNA replication from the same DNA segment during the same cell cycle. This is achieved by restricting the licensing process to G1 phase when the prereplicative complexes (preRCs) are assembled onto the origin DNA, while DNA replication is initiated only during S phase when de novo preRC assembly is blocked. Cdt1 is an important member of the preRC complex and its tight regulation through ubiquitin-dependent proteolysis and binding to its inhibitor Geminin ensure that Cdt1 will only be present in G1 phase, preventing relicensing of replication origins. We have recently reported that Cdt1 associates with chromatin in a dynamic way and recruits its inhibitor Geminin onto chromatin in vivo. Here we discuss how these dynamic Cdt1-chromatin interactions and the local recruitment of Geminin onto origins of replication by Cdt1 may provide a tight control of the licensing process in time and in space. 相似文献
3.
组蛋白乙酰化/去乙酰化作用与真核基因转录调控 总被引:1,自引:0,他引:1
核小体组蛋白的翻译后修饰是真核基因转录调控中的关键步骤。对于组蛋白的这类修饰方式 ,近年来研究最为活跃的是组蛋白N末端区域保守的Lys上ε NH 3 的乙酰化作用。随着各种组蛋白乙酰化酶 /去乙酰化酶被克隆、鉴定 ,组蛋白乙酰化 /去乙酰化作用与真核基因转录调控之间的关系也开始逐步得以阐明。1 .真核转录相关的组蛋白乙酰化酶和组蛋白去乙酰化酶1 .1 组蛋白乙酰化酶 (histoneacetyltrans ferase ,HAT) 核小体组蛋白中N末端区域上保守的Lys的乙酰化是染色质具有转录活性的标志之一。在组蛋白… 相似文献
4.
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
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Aaron C. Mason Stuart J. Haring John M. Pryor Cathy A. Staloch Tze Fei Gan Marc S. Wold 《The Journal of biological chemistry》2009,284(8):5324-5331
Replication protein A (RPA), the eukaryotic single-stranded DNA-binding
complex, is essential for multiple processes in cellular DNA metabolism. The
“canonical” RPA is composed of three subunits (RPA1, RPA2, and
RPA3); however, there is a human homolog to the RPA2 subunit, called RPA4,
that can substitute for RPA2 in complex formation. We demonstrate that the
resulting “alternative” RPA (aRPA) complex has solution and DNA
binding properties indistinguishable from the canonical RPA complex; however,
aRPA is unable to support DNA replication and inhibits canonical RPA function.
Two regions of RPA4, the putative L34 loop and the C terminus, are responsible
for inhibiting SV40 DNA replication. Given that aRPA inhibits canonical RPA
function in vitro and is found in nonproliferative tissues, these
studies indicate that RPA4 expression may prevent cellular proliferation via
replication inhibition while playing a role in maintaining the viability of
quiescent cells.Replication protein A
(RPA)3 is a stable
complex composed of three subunits (RPA1, RPA2, and RPA3) that binds
single-stranded DNA (ssDNA) nonspecifically. RPA (also referred to as
canonical RPA) is essential for cell viability
(1), and viable missense
mutations in RPA subunits can lead to defects in DNA repair pathways or show
increased chromosome instability. For example, a missense change in a high
affinity DNA-binding domain (DBD) was demonstrated to cause a high rate of
chromosome rearrangement and lymphoid tumor development in heterozygous mice
(2). RPA has also been shown to
have increased expression in colon and breast cancers
(3,
4). High RPA1 and RPA2 levels
in cancer cells are also correlated with poor overall survival
(3,
4), which is consistent with
RPA having a role in efficient cell proliferation.RPA is a highly conserved complex as all eukaryotes contain homologs of
each of the three RPA subunits
(1). At least some plants
(e.g. rice) and some protists (e.g. Cryptosporidium parvum)
contain multiple genes encoding for subunits of RPA
(5,
6). In rice, there is evidence
for multiple RPA complexes that are thought to perform different cellular
functions (5). In contrast,
only a single alternative form of RPA2, called RPA4, has been identified in
humans (7). RPA4 was originally
identified as a protein that interacts with RPA1 in a yeast two-hybrid screen
(7). The RPA4 subunit is 63%
identical/similar to RPA2. Comparison of the sequences of RPA4 and RPA2
suggests that the two proteins have a similar domain
organization.4 RPA4
appears to contain a putative core DNA-binding domain (DBD G) flanked by a
putative N-terminal phosphorylation domain and a C terminus containing a
putative winged-helix domain (Fig.
1A). The RPA4 gene is located on the X
chromosome, intronless, and found mainly in
primates.4 Initial
characterization of RPA4 by Keshav et al.
(7) indicated that either RPA2
or RPA4, but not both simultaneously, interacts with RPA1 and RPA3 to form a
complex. This analysis also showed that RPA4 is expressed in placenta and
colon tissue but was either not detected or expressed at low levels in most
established cell lines examined
(7).Open in a separate windowFIGURE 1.Properties of aRPA complex. A, schematic diagram of the
structural and functional domains of the three subunits of RPA and (proposed
for) RPA4: DNA-binding domains (DBD A-G), the phosphorylation domain
(PD), winged-helix domain (WH), and linker regions
(lines). The sequence similarity between RPA2 and RPA4 is indicated
for each domain of the subunit. B, gel analysis of 2 μg of RPA4/3,
RPA. or aRPA separated on 8-14% SDS-PAGE gels and visualized by Coomassie Blue
staining. The position of each RPA subunit is indicated. C,
hydrodynamic properties of aRPA and RPA complexes. The sedimentation
coefficient and Stokes'' radius were determined as described previously by
sedimentation on a 15-35% glycerol gradient and chromatography on a Superose 6
10/300 GL column (GE Healthcare), respectively
(13). Mass and frictional
coefficients were calculated using the method of Siegal and Monty
(8). The predicted mass was
based upon the amino acid sequence derived from the DNA sequence.These studies describe the purification and functional analysis of an
alternative RPA (aRPA) complex containing RPA1, RPA3, and RPA4. The aRPA
complex is a stable heterotrimeric complex similar in size and stability to
the canonical RPA complex (RPA1, RPA3, and RPA2). aRPA interacts with ssDNA in
a manner indistinguishable from canonical RPA; however, it does not support
DNA replication in vitro. Mixing experiments demonstrate that aRPA
also inhibits canonical RPA from functioning in DNA replication. Hybrid
protein studies paired with structural modeling have allowed for the
identification of two regions of RPA4 responsible for this inhibitory
activity. Data presented here are consistent with recent analyses of RPA4
function in human
cells,4 and we
conclude that RPA4 has anti-proliferative properties and has the potential to
play a regulatory role in human cell proliferation through the control of DNA
replication. 相似文献
9.
10.
Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts 总被引:3,自引:0,他引:3 下载免费PDF全文
Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur. 相似文献
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The procofactor, factor VIII, is activated by thrombin or factor
Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689.
The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at
either Arg-1689 or Arg-372 and influences reaction rates at these sites.
Because cleavage at Arg-372 appears rate-limiting and dependent upon initial
cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences
catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H
and R1689Q were prepared and stably expressed to slow and eliminate cleavage,
respectively. Specific activity values for the His and Gln mutations were
∼50 and ∼10%, respectively, that of wild type. Thrombin activation of
the R1689H variant showed an ∼340-fold reduction in the rate of Arg-1689
cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at
this site. Examination of heavy chain cleavages showed ∼4- and 11-fold
reductions in A2 subunit generation and ∼3- and 7-fold reductions in A1
subunit generation for the R1689H and R1689Q mutants, respectively. These
results suggest a linkage between light chain cleavage and cleavages in heavy
chain. Results obtained evaluating proteolysis of the factor VIII mutants by
factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1
subunits and in cleaving light chain at Arg-1721 from either variant,
suggesting little dependence upon prior cleavage at residue 1689 as compared
with thrombin. Overall, these results are consistent with a competition
between heavy and light chains for thrombin exosite binding and subsequent
proteolysis with binding of the former chain preferred.Factor VIII, a plasma protein missing or defective in individuals with
hemophilia A, is synthesized as an ∼300-kDa single chain polypeptide
corresponding to 2332 amino acids. Within the protein are six domains based on
internal homologies and ordered as NH2-A1-A2-B-A3-C1-C2-COOH
(1,
2). Bordering the A domains are
short segments containing high concentrations of acidic residues that follow
the A1 and A2 domains and precede the A3 domain and are designated a1
(residues 337–372), a2 (residues 711–740), and a3
(1649–1689). Factor VIII is processed by cleavage at the B-A3 junction
to generate a divalent metal ion-dependent heterodimeric protein composed of a
heavy chain (A1-a1-A2-a2-B domains) and a light chain (a3-A3-C1-C2 domains)
(3).The activated form of factor VIII, factor VIIIa, functions as a cofactor
for factor IXa, increasing its catalytic efficiency by several orders of
magnitude in the phospholipid- and Ca2+-dependent conversion of
factor X to factor Xa (4). The
factor VIII procofactor is converted to factor VIIIa through limited
proteolysis catalyzed by thrombin or factor Xa
(5,
6). Thrombin is believed to act
as the physiological activator of factor VIII, as association of factor VIII
with von Willebrand factor impairs the capacity for the membrane-dependent
factor Xa to efficiently activate the procofactor
(5,
7). Activation of factor VIII
occurs through proteolysis by either protease via cleavage of three P1
residues at Arg-740 (A2-B domain junction), Arg-372 (A1-A2 domain junction),
and Arg-1689 (a3-A3 junction)
(5). After factor VIII
activation, there is a weak electrostatic interaction between the A1 and A2
domains of factor VIIIa (8,
9) and spontaneous inactivation
of the cofactor occurs through A2 subunit dissociation from the A1/A3-C1-C2
dimer, consequently dampening factor Xase
(3).Thrombin cleavage of factor VIII appears to be an ordered pathway, with
relative rates at Arg-740 > Arg-1689 > Arg-372 and the initial
proteolysis at Arg-740 facilitating proteolysis at Arg-372 as well as Arg-1689
(10). This latter observation
was based upon results showing that mutations at Arg-740, impairing this
cleavage, significantly reduced cleavage rates at the two other P1 sites.
Thrombin-catalyzed activation of factor VIII is dependent upon interactions
involving the anion binding exosites of the proteinase
(11,
12). Exosite binding is
believed to determine substrate affinity, whereas subsequent active site
docking primarily affects Vmax
(13). Furthermore, the complex
interactions involving multiple cleavages within a single substrate may
utilize a ratcheting mechanism
(14) where presentation of the
scissile bond is facilitated by a prior cleavage event.Cleavage at Arg-372 is a critical step in thrombin activation of factor
VIII as it exposes a cryptic functional factor IXa-interactive site in the A2
domain (15), whereas cleavage
at Arg-1689 liberates factor VIII from von Willebrand factor
(16) and contributes to factor
VIIIa specific activity (17,
18). Although cleavage at
Arg-740 represents a fast step relative to cleavages at other P1 residues in
the activation of factor VIII
(19), the influence of
Arg-1689 cleavage on cleavages in the heavy chain remains unknown. In the
present study cleavage at Arg-1689 is examined using recombinant factor VIII
variants possessing single point mutations of R1689Q and R1689H. Results
indicating reduced rates of A1 and A2 subunit generation, which are dependent
upon the residue at position 1689, suggest that cleavage at Arg-1689 affects
rates of proteolysis at Arg-740 and Arg-372. These observations are consistent
with a mechanism whereby heavy chain and light chain compete for a binding
thrombin exosite(s), with heavy chain preferred over light chain. In this
competition mechanism, cleavage at Arg-740 is favored over Arg-1689.
Subsequent cleavage at Arg-372 in heavy chain may involve a ratcheting
mechanism after initial cleavage at Arg-740. On the other hand, the mechanism
for factor Xa-catalyzed activation of factor VIII appears to be less dependent
on cleavage at the Arg-1689 site as compared with thrombin. 相似文献
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Lata Balakrishnan Jason Stewart Piotr Polaczek Judith L. Campbell Robert A. Bambara 《The Journal of biological chemistry》2010,285(7):4398-4404
Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α. 相似文献