Checkpoint kinase 1 in DNA damage response and cell cycle regulation

DOI 10.1007/s00018-013-1307-3

Cellular and Molecular Life Sciences

Cell. Mol. Life Sci.

Checkpoint kinase 1 in DNA damage response and cell cycle regulation

Mallikarjun Patil · Navjotsingh Pabla · Zheng Dong

Received: 6 November 2012 / Revised: 28 January 2013 / Accepted: 18 February 2013 ? Springer Basel 2013

ATR A TM and Rad3 related Cdc25 C ell division cycle 25CDK C yclin-dependent kinases

Introduction

Checkpoint kinase 1 (Chk1) was initially identified in fis-sion yeast as a serine/threonine protein kinase that is essen-tial for DNA damage-induced cell cycle arrest [1]. Chk1 homologs were subsequently identified in other species such as Drosophila , Xenopus , mouse, and human [2–4]. Functional studies in these model systems showed that Chk1 phosphorylates the key regulators of cyclin-dependent kinase 1 (CDK1) during DNA damage, resulting in CDK1 inactivation and blockade of G2/M transition. More recent work has established important roles of Chk1 not only in DNA damage response (DDR) but also in unperturbed cell cycle. In the normal cell cycle, Chk1 mediates the check-points in S and M phases as well as G2/M transition. In this review, we discuss Chk1 regulation, its role in DDR and unperturbed cell cycle, and the possibility of targeting Chk1 for cancer therapy.

Cell cycle and DNA damage response

The cell cycle consists of a series of highly ordered, sequen-tial phases that lead to cell division [5]. A notable feature of cell cycle progression is that cells do not enter the next phase until the previous phase is completed. This feature, known as cell cycle checkpoints, provides an important sur-veillance mechanism for faithful replication and division of the cells [5] (Fig. 1). Progression from one phase of cell cycle to the next is governed by Cdks, their cyclin partners,

Abstract Originally identified as a mediator of DNA damage response (DDR), checkpoint kinase 1 (Chk1) has a broader role in checkpoint activation in DDR and normal cell cycle regulation. Chk1 activation involves phosphoryla-tion at conserved sites. However, recent work has identi-fied a splice variant of Chk1, which may regulate Chk1 in both DDR and normal cell cycle via molecular interaction. Upon activation, Chk1 phosphorylates a variety of substrate proteins, resulting in the activation of DNA damage check-points, cell cycle arrest, DNA repair, and/or cell death. Chk1 and its related signaling may be an effective therapeutic target in diseases such as cancer.

Keywords Chk1 · Cell cycle · DNA damage · Checkpoint

Abbreviations Chk1 C heckpoint kinase 1Chk2 C heckpoint kinase 2Chk1 S -checkpoint kinase 1-short DDR D NA damage response ATM A taxia telangiectasia mutated

M. Patil · N. Pabla · Z. Dong

Department of Cellular Biology and Anatomy, Georgia Regents University and Charlie Norwood v A Medical Center, 1459 Laney walker Blvd., Augusta, GA 30912, USA

Z. Dong (*)

Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha 410022, Hunan, China e-mail: zdong@https://www.sodocs.net/doc/0416957312.html,

Present Address: N. Pabla

Department of Pharmaceutical Sciences, St Jude Children’s Research Hospital, Memphis, Tennessee, USA

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protein kinases, and phosphatases [6]. Specifically, the cell cycle is driven by temporal activation of Cdks, which depends on the association of cyclin partners, phosphoryla-tion by specific protein kinases, and dephosphorylation by Cdc25 family phosphatases. For example, CDK1, the criti-cal CDK for G2/M phase transition, is activated by cyclin B binding, phosphorylation at serine-161, and dephosphoryla-tion of the inhibitory tyrosine-14 and -15 sites by Cdc25.DNA damage response is a network of signal transduc-tion pathways activated in a cell that leads to checkpoint activation, regulate cell cycle transitions, DNA repair and apoptosis in response to DNA damage [7, 8]. DNA dam-age is sensed by several molecular complexes or path-ways, the most notable of which include ATM and ATR that activate DNA damage checkpoint (Fig. 2). In general, double-strand DNA breaks are sensed by MRe11-Rad 50-Nbs1 (MRN) complex, which localizes to the DNA damage site and recruits ATM [9, 10]. ATM usually exists as homo-dimers that are inactive; however, upon localiza-tion to DNA damage sites, ATM is autophosphorylated to become monomers leading to its activation [11

]. Activated

Fig. 1 Cell cycle checkpoints. The cell cycle consists of G1, S, G2, and M phases, which are driven by various cyclin/CDK complexes. Progression from one phase to the next phase in the cell cycle is mon-itored by different checkpoints. S phase is regulated by replication checkpoint that monitors the initiation of replication, replication fork stability, fork progression, and DNA lesions. G2/M checkpoint moni-tors the completion of DNA replication with high fidelity. Spindle checkpoint makes sure that chromosomes are aligned and segregated for even distribution into two daughter cells

A B

Fig. 2 Chk1 and Chk2 activation in response to DNA damage. a Double-strand DNA (dsDNA) breaks are sensed by the MRN com-plex, which recruits and activates ATM. MRN-mediated nuclease activity generates SS DNA, which can activate ATR. ATM is also activated in Uv-induced DNA damage in ATR-dependent manner. Activated ATM phosphorylates several substrates including Chk2. RPA senses and binds single-strand DNA (ssDNA) lesions, which recruit ATR through ATRIP. 9-1-1 (Rad9-Rad1-Hus1) complex binds

to ssDNA and dsDNA junction, which recruits TOPBP1 to activate ATR, which phosphorylates Chk1. b ATR is also activated by DNA damage induced by cisplatin. Cisplatin-induced DNA lesion recruits MSH2 (mismatch repair protein 2), which in turn helps in the recruit-ment and activation of ATR. Activated ATR phosphorylates both Chk1 and Chk2, however, Chk1 is degraded and Chk2 remains active, which phosphorylates and activates p53 leading to apoptosis

Chk1 in DNA damage and cell cycle

ATM phosphorylates downstream targets including Chk2, H2AX, and MDC1 etc. [12]. Different from ATM, ATR is mainly activated by single-stand DNA generated as a result of replication stress, Uv induced DNA damage, nuclease activity consequent to double-strand breaks, enzymatic and helicase remodelling following ICL [13]. The single-strand DNA is recognized and bound by rep-lication protein A (RPA), resulting in the recruitment of ATR through its interacting partner protein called ATRIP [7, 14, 15]. Independently, RAD9-RAD1-HUS1 (9-1-1) complex binds to the ssDNA and dsDNA junction through Rad17 [16]. The 9-1-1 complex in turn helps to recruit topoisomerase binding protein 1 (TOPBP1), an activator of ATR, resulting in ATR activation [17, 18]. In addition to this canonical pathway, recent work has revealed a novel pathway of ATR activation involving mismatch repair pro-teins [19] (Fig. 2). Upon activation, ATR can phosphoryl-ate downstream targets including Chk1. ATR-mediated Chk1 phosphorylation also requires another intermediate protein called claspin, which binds and stabilizes activated Chk1. As a result, Chk1 phosphorylation was abolished in the absence of claspin in Xenopus egg extracts [20]. Although ATM and ATR are generally known to respond to double- and single-strand DNA damage respectively, their signaling pathways are not mutually exclusive. For example, double-strand breaks activate ATM, which along with MRe11 nuclease activity, generates single-strand DNA leading to consequent ATR activation [7, 21]. ATR activated in response to Uv-induced DNA damage activate ATM by phosphorylating ATM at its autophosphorylation site S1981 [22]. In addition, the downstream signaling molecules are not exclusively responsive to ATM or ATR. For example, in cisplatin-induced DNA cross-linking and replication stress, both Chk1 and Chk2 are activated in an ATR-dependent manner. while Chk1 is degraded after ini-tial activation, Chk2 phosphorylates p53 leading to its sta-bilization and activation to induce apoptosis [23]. Intertwined relationship between the cell cycle

and DDR

DNA damage response and the cell cycle are two inter-twined cellular processes. On the one hand, as discussed above, DDR leads to cell cycle arrest by activating check-point kinases. On the other hand, an emerging idea is that even the unperturbed cell cycle has a constitutive surveil-lance mechanism that is related to DDR [24, 25]. This is particularly relevant to DNA replication in S-phase, when single-strand DNA (ssDNA) and DNA breaks may be induced in several ways. During replication, template DNA is unwound at the replication fork by DNA helicases result-ing in ssDNA. ssDNA is highly susceptible to damage such as those induced by free radicals. Breaks in ssDNA at the replication fork can be converted to a double-strand break following replication. As a result, both single-strand and double-strand DNA breaks-associated DDR may be activated to slow down the progression of the cell cycle to repair the damage to ensure a faithful DNA replication and genome integrity. In this process, ATR and Chk1 play a critical role in sensing the initial ssDNA breaks to acti-vate DDR. Consistently, deficiency in either ATR or Chk1 leads to embryonic lethality in mice and embryonic stem cells of these models show cell cycle abnormalities, accu-mulate double-stand breaks and fragile site breaks [26–29]. The intertwined relationship between DDR and cell cycle provides an explanation as to why DDR proteins, such as ATR and Chk1, play such critical roles in the unperturbed cell cycle.

Chk1 in cell cycle regulation and tissue physiology Checkpoint kinase 1 was originally identified as a gene that can rescue a CDK1/cdc2 mutant in fission yeast [1]. Constitutive overexpression of Chk1 in fission yeast leads to mitotic delay, whereas loss of Chk1 has no effect on cell cycle progression. However, in the absence of Chk1, the yeast cells become more sensitive to Uv-induced DNA damage and fail to arrest in G2-phase following DNA dam-age [1]. In Drosophila, Grapes (Chk1 homolog in Drosoph-ila) regulates syncytial cell division fidelity, mitotic entry, and cyclin A degradation and as a result, it is indispensable for embryogenesis [3, 30]. In addition, Grapes may delay the accumulation of cyclin B1 in the nucleus to prevent nuclear CDK1 activation and premature entry into mitosis in Drosophila embryos [31, 32], supporting a role of Chk1 in cell cycle in early stages of embryogenesis in Drosophila. In Xenopus eggs, addition of exogenous, active Chk1 delays mitosis whereas immunodepletion of Chk1 results in an early entry into mitosis [2]. Unlike yeast, mammalian cells do not show noticeable phenotypes following Chk1 over-expression; however Chk1 knockdown and inhibition leads to reduced proliferation and cell death [33]. In mice, Chk1 knockout leads to embryonic death in utero at e6.5 stage [28, 29]. Chk1 knockout blastocysts as well as embry-onic stem cells also fail to proliferate and fail to activate the G2/M checkpoint in response to DNA damage and die shortly in culture [28, 29]. embryonic death of Chk1-null mice is independent of p53 since p53-deficiency fail to res-cue the phenotype [28]. Although viable, Chk1-deficient chicken lymphoma DT40 cells are deficient in G2 check-point in response to DNA damage and are defective in both DNA replication and cell cycle [34]. Collectively, these studies indicate that Chk1 is master regulator of cell cycle, cell survival and embryogenesis.

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In unperturbed cell cycle, Chk1 regulates DNA replication in S phase, G2/M transition or mitotic entry, and spindle checkpoint in M phase (Fig. 3). In S phase, Chk1 arrests cell cycle for DNA replication mainly by inducing Cdc25A degradation, resulting in the inhibition of CDK2. In U2OS cells, Chk1 phosphorylates Cdc25A at multiple sites and inhibition of Chk1 results in a marked accumulation of Cdc25A, suggesting that phosphorylation by Chk1 targets Cdc25A for degradation [35–37]. Consistently, conditional deletion of Chk1 from mammary cells in mice results in Cdc25A accumulation, which is accompanied by increases in S phase cells, accumulation of DNA damage, premature mitotic entry, and cell death [38]. Knockdown and inhibi-tion of Chk1 lead to increased initiation of DNA synthe-sis, increased single-strand DNA, aberrant fork structures, accumulation of double-strand breaks, DDR, and increased phosphorylation of ATR targets [33, 39]. These studies sug-gest that Chk1 may suppress late initiation of replication to prevent DNA damage for normal progression of S phase. However, the ATR/Chk1 pathway can also sense the strand stability and progression and initiate late replication to repair DNA breaks [40]. Collectively, all these observations suggest that Chk1 regulates replication checkpoint and is required for normal S phase progression and cell survival.

Mitotic entry is promoted by the activation of the cyclin B-CDK1 complex, which also involves the regulation by Chk1 [41]. Briefly, in late G2-phase, Chk1 accumulates at centrosome to phosphorylate Cdc25B (the phosphatase and activator of CDK1), resulting in the inhibition of Cdc25B and consequent suppression of CDK1. This regulation is critical to the prevention of premature mitotic entry in unperturbed cell cycle [41].

Finally, Chk1 has a critical role in spindle checkpoint in M-phase of the cell cycle. In support of this conclusion, Chk1-deficient chicken lymphoma DT 40 cells are viable, but they have increased levels of chromosome mis-segre-gation and genomic instability and fail to arrest in mitosis when treated with Taxol, a microtubule-stabilizing agent [42]. Similarly, Chk1 haploinsufficient primary mammary epithelial cells also exhibit misaligned chromosomes, chromosome missegregation, and enhanced binucleation [43]. Checkpoint kinase 1 knockdown U2OS cells undergo aberrant mitosis and fail to activate spindle checkpoint in response to Nocodazole treatment [37]. These studies suggest defective spindle checkpoint in the absence of Chk1. Mechanistically, Chk1 regulates spindle checkpoint through the activation and localization of Aurora B kinase, a regulator of spindle checkpoint that recruits BubR1 and MAD1 to the kinetochores [42, 43] to prevent APC/C acti-vation and delay anaphase onset [44]. Chk1 deficiency leads to mislocalization of Aurora B, BubR1 and MAD1 proteins, resulting in spindle checkpoint failure [42, 43] (Fig. 3).

Chk1 in DNA damage response

Originally identified in yeast, Chk1 is now recognized as an important mediator and signal transducer in DDR in other eukaryotes including mammals. In response to DNA damage, Chk1 is rapidly phosphorylated at serine-317 and serine-345 by ATR and becomes highly activated, resulting in the activation of DNA damage checkpoints [28, 45, 46]. Activated Chk1 phosphorylates several downstream targets to bring about cell cycle arrest, activate DNA repair path-ways, and induce apoptosis when DNA damage is severe [8, 47–49].

Chk1-induced cell cycle arrest in DDR

Checkpoint kinase 1 induces cell cycle arrest in DDR mainly by phosphorylating Cdc25 family phosphatases, wee1 kinase and controlling PLK1 (Fig. 4). The Cdc25 family is dual-specificity phosphatases that can dephospho-rylate Cdks in their ATP-binding loop, resulting in the acti-vation of Cdks and cell cycle progression. In mammalian

cells, there are three (A, B and C) isoforms of Cdc25, all

Fig. 3 Cell cycle checkpoints regulated by Chk1. Chk1 helps main-tain genomic integrity by regulating DNA replication, G2/M, and spindle checkpoints. Chk1 monitors DNA replication, slows down the replication to favor fork stability, prevent stalling of forks and DNA breaks. Chk1 prevents premature entry into mitosis before DNA replication is completed with high fidelity by inhibiting Cdc25 fam-ily phosphatases. Chk1 also regulates segregation of chromosomes through activation of aurora kinase to prevent genomic instability

Chk1 in DNA damage and cell cycle

of which can be phosphorylated by Chk1 in DDR. Interest-ingly, upon phosphorylation Cdc25A, B and C may employ distinct mechanisms to arrest the cell cycle [50, 51] (Fig. 5). For Cdc25A, phosphorylation by Chk1 targets it for protea-somal degradation, which leads to the inhibition of Cdk1 and Cdk2 resulting in cell cycle arrest at G1/S transition, S

phase, and G2/M transition [52, 53]. Interestingly, besides direct phosphorylation, Chk1 can also activate Nek11, which in turn phosphorylates Cdc25A to induce cell cycle arrest during DDR [54]. For Cdc25C, a main regulation seems to be its cellular localization. Specifically, phosphorylation of Cdc25C by Chk1 promotes its binding to 14-3-3 proteins leading to the sequestration of Cdc25C from Cdk1, Cdk1 inactivation and G2 arrest [4]. Cdc25B regulation by Chk1 seems to occur at centrosomes, where Chk1 phosphorylates Cdc25B leading to its sequestration from centrosome and inhibition of centrosomal Cdk1 [41].

In addition to Cdc25 phosphotases, Chk1 phosphoryl-ates wee1, the protein kinase that is responsible for the inhibitory phosphorylation of Cdk1 (Fig. 4). As a result, in response to DNA damage, the phosphorylation and activa-tion of wee1 by Chk1 leads to the inhibition of Cdk1 and cell cycle arrest at G2 phase [55, 56]. Chk1 is also a nega-tive regulator of PLK1 (polo like kinase 1), a mitotic kinase involved in centrosome maturation, spindle formation, and cytokinesis [57]. PLK1 phosphorylates wee1 and targets it for degradation, leading to Cdk1 activation and mitotic entry (Fig. 4). In addition, recent work suggests that, in response to DNA damage, Chk1 dissociates from the chro-matin leading to decreased phosphorylation and acetyla-tion of histone H3, which may contribute to DNA damage-associated transcriptional repression [58]. Interestingly, the repressed genes include cyclin B and Cdk1, although the role of the transcriptional repression in cell cycle arrest remains unclear.

Chk1-mediated DNA repair during DDR

As discussed above, Chk1 induces checkpoint activa-tion resulting in cell cycle arrest. In addition, in S-phase, Chk1 may slow down DNA replication by blocking the ini-tiation factor CDC45 in a Cdk2-independent manner [59]. Together, these mechanisms provide time for DNA repair to maintain genomic integrity in response to DNA damage. Notably, emerging evidence from the past few years has suggested a more direct role of Chk1 in DNA repair (Fig. 6).In this regard, Chk1 induces post-translational modifica-tion and activation of several important DNA repair factors. (1) Proliferating cell nuclear antigen (PCNA). One of the mechanisms of Chk1-mediated DNA repair is by inducing monoubiquitination of PCNA [60]. Upon ubiquitination, PCNA can activate translesion synthesis, a process that allows the replication machinery to pass or tolerate DNA lesions for replication. Specifically, at DNA damage sites, monoubiquitinated PCNA recruits specific translesion syn-thesis DNA polymerases to replace classical polymerases [61, 62], which can bypass or repair the damaged sites (lesion) for DNA replication [63]. (2) FANCe. In the repair

of DNA cross-links, Chk1 phosphorylates the FANCe

Fig. 4 Chk1 regulation of cell cycle progression. Chk1 regulates pro-gression of cell cycle by inhibiting Cdc25 family phosphatases and polo-like kinase 1(PLK1), and activating wee 1 kinase. Cdc25 is a phosphatase and activator of CDK1, whereas wee1 inhibits CDK1 by phosphorylation. PLK1 can activate CDK1 by inhibiting wee1.

PLK1 can also directly promote cell cycle progression

Fig. 5 Chk1-mediated cell cycle arrest in response to DNA dam-age. Chk1 phosphorylates Cdc25 family resulting in their inhibition. Chk1 can also activate Nek11, which further phosphorylates Cdc25A on multiple sites to target it for degradation. Cdc25A is crucial for G1-S transition, S phase progression, and mitotic entry; as a result, Cdc25A inhibition leads to G1 arrest, slows down replication, and G2 arrest. Chk1 phosphorylates Cdc25B to promote its binding to 14-3-3 protein and sequestration from centrosome. Chk1 also phosphorylates Cdc25C to induce its binding to 14-3-3 protein and nuclear exclusion. Cdc25B and Cdc25C inhibition mainly causes G2 arrest

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subunit of the Fanconi anemia (FA) core complex on two conserved sites serine-346 and serine-S374, resulting in its co-localization with FANCD2 at nuclear foci or DNA dam-age sites [44, 64]. Together with BRCA1 and 2, FANCe and ubiquitinated FANCD2 help resolve interstrand DNA crosslinks [44]. (3) Rad51. Checkpoint kinase 1 participates in homologous recombination repair by regulating Rad51. Knockdown or inhibition of Chk1 leads to accumulation of double-strand breaks and increased cell death in response to double-strand breaks induced by hydroxyurea and camp-tothecin. Mechanistically, Chk1 phosphorylates Rad51 at serine-309 and recruits it to DNA repair foci to promote homologous recombination repair and cell survival [65]. (4) TLK. In response to double-strand DNA breaks, Chk1 can also phosphorylate the serine-695 site of TLK (tousled-like kinase), an evolutionarily conserved serine/threonine kinase that is involved in chromatin assembly, DNA replica-tion, and repair. Interestingly, the phosphorylation of TLK by Chk1 depends on ATM [66]. Finally, it is noteworthy that Chk1 has also been suggested to stabilize replication forks and/or restart stalled replication forks; however, the under-lying mechanisms remain unclear.Chk1-induced cell death in DDR

There are few reports implicating Chk1 in apoptosis or cell death. Chk1 phosphorylates p73 at serine-47, resulting in a

drastic increase in p73 expression and transcription activity in response to DNA damage. Although p73 is a p53-related protein, it induces apoptosis independently of p53 [67, 68]. In contrast, there are instances where Chk1 inhibits apopto-sis through ATM/ATR-caspase2 and RPA-caspase3 pathway [69, 70]. Therefore, it remains unclear whether (and to what extent) Chk1 contributes to apoptosis and its regulation.

Regulation of Chk1

At the sequence level, Chk1 is highly conserved from yeast to humans [52] (Fig. 7

). Structurally, Chk1 has a conserved N-terminal kinase domain, a linker region, a regulatory SQ/TQ domain, and a C-terminal domain with unclear func-tions [52, 71]. It is noteworthy that Chk1 and Chk2 are not homologous in sequence, although they both have check-point function and share some phosphorylation substrate proteins.

It is well recognized that Chk1 is activated upon phospho-rylation at two conserved sites, serine-317 and serine-345 [28, 46, 72]. Although phosphorylation at these two sites is indispensable for the function of Chk1, it remains elu-sive as to how exactly the phosphorylation activates Chk1 [45]. The study of crystallographic structure revealed that in the absence of phosphorylation, the Chk1 kinase domain assumes an open, active conformation [73]. Indeed, the

Fig. 6 Chk1-mediated DNA repair. Activated Chk1 phosphorylates several substrates to mediate DNA repair and apoptosis. Chk1 slows down DNA replication directly by affecting CDC45 loading on replica-tion complex to allow time for DNA repair. Chk1 promotes monoubiq-uitination of PCNA, which recruits translesion polymerases to help translesion DNA synthesis. Chk1 phosphorylates TLK-tousled like kinase to promote chromatin assembly. Chk1 phosphorylates Rad51 to target it to double-strand breaks to promote HRR-homologous

recombination and repair. Chk1 phosphorylates FANCe and mon-oubiquitinates FANCD2, which help resolve ICR-interstrand cross-link DNA lesion. Chk1 also regulates replication fork stability, fork

restart, and late origins of replication, the mechanistic details of which are currently unclear. In addition to DNA repair, Chk1 may also induce cell cycle arrest and apoptosis by regulating different sub-strates

Chk1 in DNA damage and cell cycle kinase domain has significantly higher kinase activity when compared to full-length Chk1. These observations indicate that Chk1 kinase domain has a constitutively active confor-mation that does not rely on phosphorylation for activation. One model of Chk1 activation proposes that normally the C-terminal domain interacts with the kinase domain to mask the active site and the phosphorylation at serine-317 and ser-ine-345 dissociates these two domains leading to Chk1 acti-vation. This model is supported by several lines of evidence. First, C-terminal truncation mutants show higher catalytic activity than intact Chk1 [73]. Second, in binding assays, ectopically expressed C-terminal domain of rat Chk1 binds to the N-terminal kinase domain [74]. In addition, in Xeno-pus egg extracts, ectopically expressed C-terminal domain can interact with N-terminal Chk1 domain and inhibit its activity but not full-length Chk1 [71]. The inhibitory effect of C-terminal Chk1 domain on N-terminal kinase domain can be overcome by treatment with aphidicolin and phos-phomimetic mutations of ATR [71], suggesting that the C-terminal domain indeed inhibits kinase activity. Together, these observations suggest that the C-terminal domain is inhibitory to the kinase domain in Chk1 and, upon phos-phorylation, this inhibition is disabled. while this “intra-molecular inhibition” model is supported by several lines of evidence, it has been challenged recently. For exam-ple, several C-terminal truncation mutants lead to loss of function in Chk1 [45, 75], suggesting that removal of the C-terminal domain is not sufficient for Chk1 activation and Chk1 regulation may be more complex than “intramolecular

inhibition”. In addition to serine-317 and serine-345, Chk1

Fig. 7 CHk1 sequence align-ment between human, mice, fruit fly, Xenopus and yeast. Chk1 sequences of human, mice, Drosophila , Xenopus , and yeast were analyzed using T-coffee, a web server for mul-tiple sequence alignment tool. The N-terminal kinase domain and regulatory SQ/TQ domain of Chk1 are highly conserved, while the C-terminal domain is less conserved. Asterisk indicates highly conserved sequence, Colon indicates con-served substitution, Dot indicate semi conserved substitutions

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has also been reported to have autophosphorylation at ser-ine-296, which plays a role in G2 checkpoint [76]. Interest-ingly, the autophosphorylation is consequent to phosphoryl-ation at serine-317 and serine-345 through intramolecular mechanisms. It is noteworthy that serine-296 phosphoryla-tion does not affect the kinase activity of Chk1 [77].

Interestingly, a recent study by walker and colleagues indicates that Chk1 may be subjected to a new mechanism of regulation, called “de-repression”. In this model, Chk1 is antagonized by an inhibitory or repressing factor(s), the dissociation of which leads to Chk1 activation. In support of this possibility, Chk1 immunoprecipitated from untreated cells demonstrates significantly higher kinase activity after being washed with a more stringent buffer. Notably, Chk1 immunoprecipitated from DNA damaged (by aphidicolin) cells also shows significantly higher kinase activity when washed with the stringent buffer, although the increase is somewhat smaller than that of untreated cells. In addition, stringent buffer washes also increase the kinase activity of Chk1 (S345A) mutant from transfected cells [78], suggest-ing that phosphorylation at serine-345 is not obligatory for Chk1 kinase activity per se, although it plays a role in Chk1

activation in cells. Furthermore, Chk1 can be activated in a claspin-dependent manner in response to genotoxic stress independently of ATM/ATR and associated phosphoryla-tion [79]. Together, these studies support the possibility of additional regulatory mechanisms for Chk1, in addition to phosphorylation. The “de-repression” model is intriguing and tantalizing, but the identity of the “repressing” factor(s) of Chk1 is not known.

Our latest work has identified a splice variant of Chk1, which may act as an endogenous repressor of Chk1 [80]. This variant is spliced in a way that the exon 3 of Chk1 is deleted, leading to the production of an N-terminally trun-cated protein, which we call Chk1-short or Chk1-S. Due to the truncation, Chk1-S lacks part of the kinase domain including the ATP-binding (Fig. 8a) and, as expected, does not possess protein kinase activity. Importantly, a series of in vitro and in vivo tests showed that Chk1-S directly interacts with Chk1 at its N-terminal domain and inhibits Chk1 kinase activity. In addition, we confirmed the find-ing by walker and colleagues that stringent buffer washes can increase the activity of Chk1 immunoprecipitated from

cells. Moreover, we showed that the addition of exogenous

Fig. 8 Physical and functional interactions between Chk1 and Chk1-S. a Schematic representation of Chk1 and Chk1S domains and their interaction. Checkpoint kinase 1 has ATP binding domain, N-terminal kinase domain, SQ/TQ domain, and C-terminal domain. Chk1S lacks ATP binding domain. C-terminal domain of Chk1 inter-acts with N-terminal domain of CHK1-S. b Regulation of Chk1 by Chk1-S. Chk1-S interacts with Chk1 to inhibit its kinase activity. In unperturbed cell cycles, Chk1-S is expressed in late S to G2 phase to inhibit Chk1 and promote mitotic entry. In DNA damage, Chk1 is phosphorylated to prevent Chk1-S binding and inhibition, result-ing in Chk1 activation. when Chk1-S is overexpressed, it antagonizes Chk1, leading to premature mitotic entry and consequent cell death via mitotic catastrophe. Figure 8b is adopted from Pabla et al. PNAS 109, 197-202, 2012

Chk1 in DNA damage and cell cycle

Chk1-S can reverse the effect of stringent buffer washing. Together, these results suggest that Chk1-S may be one of the key endogenous repressing factors of Chk1. In an unper-turbed cell cycle, Chk1-S expression increases significantly in late G2 and early M phases coinciding with the decrease in Chk1 activity for mitotic entry. Overexpression of Chk1-S leads to premature mitotic entry followed by cell death in the form of mitotic catastrophe. These observations sup-port an important role of Chk1-S in the regulation of normal cell cycles by binding and antagonizing Chk1. In response to DNA damage, the interaction between Chk1 and Chk1-S is lost in an ATR-dependent manner (Fig. 8b). Remarkably, mutation of Chk1 at serine-317 and serine-345 prevents the dissociation of Chk1 from Chk1-S during DNA dam-age, indicating that the dissociation of Chk1 from Chk1-S in DDR depends on Chk1 phosphorylation. Based on these observations, we propose that Chk1-S is a key regulator of Chk1 in both normal cell cycles and DDR. Mechanisti-cally, Chk1-S binds to Chk1 to act as a repressor to block Chk1 activity. In normal cell cycles, Chk1-S is drastically increased at late G2 phase to repress Chk1 to promote mitotic entry. In DDR, ATR-mediated Chk1 phosphoryla-tion at serine-317 and serine-345 leads to the dissociation of Chk1-S from Chk1, resulting in Chk1 activation and cell cycle arrest (Fig. 8b).

In addition to the above-described regulatory mecha-nisms, Chk1 is also known to be subjected by proteasomal degradation. Interestingly, proteasomal degradation of Chk1 may depend on its phosphorylation status. In camptothecin-induced DNA damage, Chk1 phosphorylation at serine-345 targets the protein for proteosomal degradation [81]. It was later shown that the phosphorylation exposes a dragon-like region at the C-terminus, which is recognized by the F-box protein called Fbx6, leading to Chk1 ubiquitination and proteasomal degradation and termination of the checkpoint [82]. Consistently, there is an inverse correlation between Chk1 and Fbx6 expression in cultured cancer cells and breast cancer tissues. In addition, overexpression of Fbx 6 leads to degradation of chk1 and increased cellular sensitiv-ity to camptothecin [82].

Chk1 substrates

Despite the multiple roles of Chk1 in normal cell cycles and DDR, only a few phosphorylation substrates of Chk1 have been identified thus far. As a result, identification of func-tional substrates of Chk1 is currently a subject of intense investigation. The consensus sequence of Chk1-specific sub-strates has been suggested by analyzing the peptide derived from Xenopus. Cdc25C containing serine-287, a typical Chk1-phosphorylation site. Indeed, the consensus sequence has been useful for predicting Chk1 substrates including PDS1 (an anaphase inhibitor) and wee1 [83]. Nonetheless, the consensus sequence is insufficient to explain other Chk1 substrates. Some of the Chk1 substrates may be context-dependent and do not rely on the consensus sequence for phosphorylation. In this regard, a substrate may be phospho-rylated by Chk1 through interaction with adaptor proteins.

A recent study has conducted a global phospho-proteomic screen using analogue-sensitive Chk1 and suggested 171 proteins as its potential phosphorylation substrates. Moreo-ver, KAP1, a protein phosphorylated in response to DNA damage, has been validated to be a Chk1 substrate in both in vitro and in vivo studies and KAP1 phosphorylation can be used as a read out for Chk1 activation [47]. However, most of the substrates implied in this study remain to be validated. It is expected that new and important functional substrates of Chk1 will be discovered in the next few years to provide new insights into the multiple functions of Chk1 in the cell cycle and DDR.

Chk1 in cancer

Checkpoint kinase 1 mutations or loss are very rare in can-cer. A logical explanation is that Chk1 plays an essential role in cell cycle regulation, cell proliferation and survival [28, 29, 80], and as a result, cells with defective Chk1 are eliminated during tumorigenesis. Nonetheless, par-tial ablation of Chk1 (heterozygous) favors tumor forma-tion in wNT1 oncogenic mice [28]. In addition, loss-of-function Chk1 mutations have been reported in stomach, endometrial, and colorectal cancers [84–87]. Mutations have been mapped to the microsatellite instability region in the coding region of Chk1 with a stretch of nine adenine nucleotides [86]. Frame-shift mutations lead to the forma-tion of a truncated protein in colorectal and endometrial cancers lacking part of the kinase domain and the C-ter-minal domain [86]. Intriguingly, in these cancers is that the second allele is normal, resulting in the expression of functional Chk1 albeit at a lower level than that of normal tissues [88]. Monoallelic mutations leading to partial loss of expression are also observed in ATR in endometrial and stomach cancers [85]. These studies support the haploin-sufficient tumor model in which monoallelic mutations in several genes of the same pathway favor tumorigenesis [88]. In mice, conditional deletion of Chk1 in mammary epithelial cells fails to produce tumors; instead it leads to cell death [38]. However, the mice haploinsufficient for both Chk1 and p53 develop tumors in mammary glands [89]. Similarly, complete loss of Chk1 in skin suppresses chemically induced carcinogenesis, whereas partial (hap-loinsufficient) deletion of Chk1 promotes benign malig-nant tumor progression [90]. Triple-negative (estrogen-/ progesterone-/HeR2-) breast cancers express a very high

M. Patil et al.

level of Chk1 and have poor clinical outcomes, suggest-ing that Chk1 favors cell proliferation [91]. Together, these studies indicate that Chk1 is essential for cell survival and its haploinsufficiency may promote cancer, especially in the presence of mutations of other relevant genes. Of note, Chk1-S, the splice variant and endogenous inhibitor of Chk1, is expressed at significantly higher levels in cancer tissues than normal tissues and notably, Chk1-S expres-sion correlates with the degree of malignancy in ovarian and testicular tumors [80]. Interestingly, Chk1 expression is also higher in cancer. It is suggested that relatively high levels of Chk1 and Chk1-S may favor cell proliferation in cancer.

Targeting Chk1 for cancer therapy

DNA damaging agents are the most commonly used drugs for cancer therapy. By damaging DNA, these chemother-apy drugs induce cell cycle arrest to prevent cell prolifera-tion and trigger cell death in cancers. Many of these agents induce G1 arrest in a p53-dependent manner and G2 arrest in a Chk1-dependent manner. G1 arrest is defective in many cancers since more than 50 % of cancers are defective in p53; under these situations, G2 arrest becomes a main path-way for cancer cell survival. Hence, Chk1-dependent G2 checkpoint is a major target for chemotherapy [92–95]. In this regard, Chk1 inhibition or knockdown increases the sensitivity of p53-deficient cancer cells to DNA damage agents [33, 96]. However, it has also been reported in certain cell lines that Chk1 is essential for G2 checkpoint irrespec-tive of the p53 status and hence Chk1 inhibition may not preferantially kill p53-deficient cells [97]. It has also been shown that Chk1 inhibition increases the sensitivity of can-cer cells to anti-mitotic agents [98, 99]. In addition, Chk1 is a client of HSP90 and inhibition of HSP90 leads to the loss of Chk1 resulting in the sensitization of cancer cells to Gemcitabine, an S phase chemotherapeutic agent [100]. Chk1 and wee1 kinase inhibitors in combination have more than additive effect on different cancer cell line prolifera-tion and human xenograft models [101]. Therefore, Chk1 inhibitors may target multiple cell cycle phases to override the checkpoint responses during cancer therapy, leading to increased therapeutic efficacy. Currently, Chk1 inhibitors are being developed and tested alone or in combination with DNA damaging agents in clinical trials for cancer therapy [8, 36]. However, so far, none of these have passed phase III clinical trials. Obviously, the specificity and potency of Chk1 inhibitors are critical to their clinical use. In addition, their side effects have to be carefully evaluated because Chk1 has an important role in cell cycle regulation and its inhibition may adversely affect cell viability and function in normal tissues.Concluding remarks

Checkpoint kinase 1 plays an essential role in cell cycle regulation and DNA damage response. In unperturbed cell cycle, Chk1 regulates G1/S transition, S phase, mitotic entry, and mitosis. In DDR, Chk1 is an important signal transducer and the trigger of G2 checkpoint activation. The role of Chk1 in unperturbed cell cycle and tissue physiol-ogy is only beginning to be understood. Chk1 regulates S phase progression by controlling DNA replication, fork sta-bility and late origins of replication the mechanistic details of which are yet to be identified. The role of Chk1 in the development and pathogenesis in different tissues are yet to be investigated. In addition, although Chk1 regulates sev-eral checkpoints, only a few Chk1 substrate proteins have been identified. The discovery of Chk1-S as an important regulator of Chk1 in both unperturbed cell cycle and DDR has opened new areas of investigation. Finally, Chk1 may be an effective therapeutic target in diseases. In this regard, Chk1 inhibitors, when used together with other therapeutic agents, may significantly enhance the chemotherapy effi-cacy in cancer treatment.

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tumor cells to replication stress. J Biol Chem 278:52572–52577 101. Guertin AD, Martin MM, Roberts B, Hurd M, Qu X, Miselis NR, Liu Y, Li J, Feldman I, Benita Y, Bloecher A, Toniatti C, Shumway SD (2012) Unique functions of CHK1 and wee1 underlie synergistic anti-tumor activity upon pharmacologic inhibition. Cancer cell Int 12:45

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状态检测 / FireWall-1 1993 OPSEC 1997 VPN-1 1998 Next Generation 2001 SmartDefense 2002 应用智能 2003 Check Point: 一直走在客户现实需求的前面 创新历程 1994 1995 1996 1999 2000 Web 智能 2004

我们的策略 2004上半年提供! ? 安全远程访问 ? Web 服务器保护 ? 统一认证 ? 一致性策略管理 ? 市场领先 ? 十年的成功史 ? 最新发布 - InterSpect 2.0 - Connectra 2.0 - Intergrity 6.0 ? Check Point InterSpect ? Zone Labs SMART 管理 无忧保护 边界 深入检查 智能 安全解决方案

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本篇要为大家介绍一些实用的知识，那就是如何配置防火中的安全策略。但要注意的是，防火墙的具体配置方法也不是千篇一律的，不要说不同品牌，就是同一品牌的不同型号也不完全一样，所以在此也只能对一些通用防火墙配置方法作一基本介绍。同时，具体的防火墙策略配置会因具体的应用环境不同而有较大区别。首先介绍一些基本的配置原则。 一. 防火墙的基本配置原则 默认情况下，所有的防火墙都是按以下两种情况配置的： ?拒绝所有的流量，这需要在你的网络中特殊指定能够进入和出去的流量的一些类型。 ?允许所有的流量，这种情况需要你特殊指定要拒绝的流量的类型。可论证地，大多数防火墙默认都是拒绝所有的流量作为安全选项。一旦你安装防火墙后，你需要打开一些必要的端口来使防火墙内的用户在通过验证之后可以访问系统。换句话说，如果你想让你的员工们能够发送和接收Email，你必须在防火墙上设置相应的规则或开启允许POP3 和SMTP 的进程。 在防火墙的配置中，我们首先要遵循的原则就是安全实用，从这个角度考虑，在防火墙的配置过程中需坚持以下三个基本原则：（1）. 简单实用：对防火墙环境设计来讲，首要的就是越简单越好。其实这也是任何事物的基本原则。越简单的实现方式，越容易理解和使用。而且是设计越简单，越不容易出错，防火墙的安全功能越容易得到保证，管理也越可靠和简便。 每种产品在开发前都会有其主要功能定位，比如防火墙产品的初衷就是实现网络之间的安全控制，入侵检测产品主要针对网络非法行为进行监控。但是随着技术的成熟和发展，这些产品在原来的主要功能之外或多或少地增加了一些增值功能，比如在防火墙上增加了查杀病毒、入侵检测等功能，在入侵检测上增加了病毒查杀功能。但是这些增值功能并不是所有应用环境都需要，在配置时我们也可针对具体应用环境进行配置，不必要对每一功能都详细配置，这样一则会大大增强配置难度，同时还可能因各方面配置不协调，引起新的安全漏洞，得不偿失。 （2）. 全面深入：单一的防御措施是难以保障系统的安全的，只有采用全面的、多层次的深层防御战略体系才能实现系统的真正安全。在防火墙配置中，我们不要停留在几个表面的防火墙语句上，而应系统地看等整个网络的安全防护体系，尽量使各方面的配置相互加强，从深层次上防护整个系统。这方面可以体现在两个方面：一方面体现在防火墙系统的部署上，多层次的防火墙部署体系，即采用集互联网边界防火墙、部门边界防火墙和主机防火墙于一体的层次防御；另一方面将入侵检测、网络加密、病毒查杀等多种安全措施结合在一起的多层安全体系。 3）. 内外兼顾：防火墙的一个特点是防外不防内，其实在现实的网络环境

Checkpoint防火墙测试用例

Checkpoint R65 Test Plan Revision 0.1 2009-10-14 Author: Peng Gong

Revision History

1‘FW MONITOR’ USAGE (7) 2TEST SUITES (7) 2.1R65I NSTALL &U NINSTALL (7) 2.1.1Create a v3 vap-group with vap-count/max-load-count set >1, Confirm R65 and related HFA could be installed on all vap within vap-group successfully (7) 2.1.2Create a v4 vap-group, confirm XOS would prompt correctly message and failure install would cause unexpected issue to system (9) 2.1.3Create a v5 vap-group, confirm XOS would prompt correctly message and failure install would cause unexpected issue to system (10) 2.1.4Create a v5_64 vap-group, confirm XOS would prompt correctly message and failure install would cause unexpected issue to system (11) 2.1.5Try to install R65 on a v3 vap-group and abort the install process, confirm nothing is left afterwards12 2.1.6Reduce max-load-count down to 1, confirm only one Vap is allowed to run R65 accordingly (13) 2.1.7Increase vap-count and max-load-count to maximum vap count and executing "application-update", confirm all the Vap are running R65 properly (14) 2.1.8Reduce vap-count to 3, Confirm the surplus Vap is stopped from running R65 and related partitions are removed from CPM (15) 2.1.9Enable/Disable IPv6 in vap-group and w/o ipv6-forwarding, confirm R65 wouldn't be affected. (16) 2.1.10Confirm the installed R65 could be stopped through CLI (17) 2.1.11Confirm the installed R65 could be started through CLI (18) 2.1.12Confirm the installed R65 could be restarted through CLI (18) 2.1.13Confirm the installed R65 could be reconfigured through CLI (19) 2.1.14Confirm checkpoint could be upgraded to R70 by CLI: application-upgrade (19) 2.1.15Confirm R65 could run properly while reload vap-group totally (20) 2.1.16Confirm R65 could run properly while reload all chassis totally (20) 2.1.17Create 2 vap groups, install R65 on both. Confirm R65 are running on both vap-groups without any mutual impact. (21) 2.1.18Confirm configure operation couldn't be allowed if some Vap don't run R65 properly. (23) 2.1.19Confirm uninstall/Configure operation couldn't be allowed if some Vap don't run properly. (24) 2.1.20Confirm start/stop/restart operation could works prope rly if R65 doesn’t run properly on some Vaps.25 2.1.21Confirm configure operation couldn't be allowed if vap-count>max-load-count causing some Vap don't run properly (26) 2.1.22Confirm uninstall operation couldn't be allowed if vap-count>max-load-count causing some Vap don't run properly (27) 2.1.23Confirm start/stop/restart operation couldn't be allowed if vap-count>max-load-count causing some Vap don't run properly (28) 2.1.24Confirm R65 could be uninstalled from vap-group (29) 2.1.25Confirm R65 installation package could be remove from XOS by CLI: application-remove (30) 2.2C HECKPOINT F EATURES (31) 2.2.1Create GW and Set SIC, get topology from Smart Dashboard (31) 2.2.2Configure Policy and push policy (31) 2.2.3Confirm R65 could be started/stoped/restarted by cp-command (32) 2.2.4Confirm different kinds of rule actions could work - allow/drop/reject (33) 2.2.5Verify different kinds of tracking actions could work properly - log/alert/account (33) 2.2.6Verify static NAT and hide NAT work properly (34) 2.2.7Verify default license is installed automatically (34) 2.2.8Verify some usual fw commands output - fw ver -k/ fw ctl pstat/ fw tab/ fw stat/ cphaprob stat /fw fetch /fw unloadlocal / fwaccel stat (34) 2.2.9Verify SXL status if enable/disable it (36) 2.2.10Verify the log information in SmartViewer are displayed correctly. (37) 2.2.11Verify the GW and its member information in SmartViewer are displayed correctly (37) 2.2.12Define a VPN Community between two GW; Confirm traffic can pass (37) 2.2.13Create a VPN client tunnel, make sure client can login and traffic is encrypted as it should (38) 2.2.14Enabling/Disabling SXL during connections (39) 2.2.15Fail over the active member during connections (39) 2.3I NTERFACE T EST-T RAFFIC MIXED WITH IPV6 AND IPV4 (39) 2.3.1Verify traffic pass through the interface without SXL - non vlan<--->vlan (tcp+udp+icmp) (39)

第一讲燃气轮机基本原理及9E燃机性能型号参数

第一讲：燃气轮机基本原理及9E燃机性能型号参数授课内容： 第一章：绪论 1）：燃气轮机发电装置的组成 2）：燃气轮机发展史 3）：我国燃气轮机工业慨况 4）：GE公司燃气轮机产品系列及其编号 第二章：燃气轮机热力学基础知识 1）：工质的状态参数 2）：理想气体状态方程 3）：功和热量 第三章：燃气轮机热力循环 1）：燃气轮机热力循环的主要技术指标 2）：燃气轮机理想简单循环 3）：燃气—蒸汽联合循环 第四章：9E燃机性能型号参数 1）：PG9171E型燃机型号简介 2）：PG9171E型燃机性能参数简介

第一章绪论 第一节燃气轮机发电装置的组成 燃气轮机是近几十年迅速发展起来的热能动力机械。现广泛应用的是按开式循环工作的燃气轮机。它不断地由外界吸入空气，经过压气机压缩，在燃烧室中通过与燃料混合燃烧加热，产生具有较高压力的高温燃气，再进入透平膨胀作功，并把废气排入大气。输出的机械功可作为驱动动力之用。因此，由压气机、燃烧室、透平再加上控制系统及基本的辅助设备，就组成了燃气轮机装置。如果用以驱动发电机供应电力，就成了燃气轮机发电装置。 (幻灯)

第二节 燃气轮机发展史 燃气轮机是继汽轮机和内燃机问世以后，吸取了二者之长而设计出来的，它

是内燃的，避免了汽轮机需要庞大锅炉的缺点;又是回转式的，免去了内燃机中将往复式运动转换成旋转运动而带来的结构复杂，磨损件多，运转不平稳等缺点。但由于燃气轮机对空气动力学和高温材料的要求超过其他动力机械，因此，发展燃气轮机并使之实用化，人们为之奋斗了很长时间。如果从1791年英国人约翰·巴贝尔(John Baber)申请登记第一个燃气轮机设计专利算起，经过了半个世纪的奋斗，到1939年，一台用于电站发电的燃气轮机(400OkW)才由瑞士BBC公司制成，正式投运。同时Heinkel工厂的第一台涡轮喷气式发动机试飞成功，这标志着燃气轮机发展成熟而进入了实用阶段·在此以后，燃气轮机的发展是很迅速的。由于燃气轮机本身固有的优点和其技术经济性能的不断提高，它的应用很快地扩展到了国民经济的很多部门· 首先在石油工业中，由于油田的开发和建设，用电量急剧增加·建造大功率烧煤电站不具备条件(没有煤炭，交通不便，水源紧张，施工困难等)，周期也不能满足要求·而燃气轮机电厂功率不受限制，建造速度抉，对现场条件要求不高，油田有充足的可供燃用的气体和液体燃料·不少油田还利用开发过程中一时难以利用的伴生气作燃气轮机燃料，价格便宜，发电成本低，增加了燃气轮机的竞争力，所以在油田地区，燃气轮机装置被广泛应用，除用于发电外，还在多种生产作业申用燃气轮机带动压缩机(例如天然气管道输送，天然气回注，气田采油等)和泵(例如原油管道输送和注水等)。 其他工业部门，如炼油厂、石油化工厂、化工厂、造纸厂等等;它们不仅需要机械动力，而且需要大量热(例如蒸汽)。这时用燃气轮机来功热联供，在满足这两方面需要的同时，还能有效地节能，故应用发展较快。 实践证明，燃气轮机作为舰船推进动力，其优点显著，特别是排水量为数千

防火墙基础知识

防火墙基础知识 3.3 包过滤包过滤技术(Ip Filtering or packet filtering) 的原理在于监视并过滤网络上流入流出的Ip包，拒绝发送可疑的包。由于Internet 与Intranet 的连接多数都要使用路由器，所以Router成为内外通信的必经端口，Router的厂商在Router上加入IP Filtering 功能，这样的Router也就成为Screening Router 或称为Circuit-level gateway. 网络专家Steven.M.Bellovin认为这种Firewall 应该是足够安全的，但前提是配置合理。然而一个包过滤规则是否完全严密及必要是很难判定的，因而在安全要求较高的场合，通常还配合使用其它的技术来加强安全性。Router 逐一审查每份数据包以判定它是否与其它包过滤规则相匹配。(注：只检查包头的内容，不理会包内的正文信息内容) 过滤规则以用于IP顺行处理的包头信息为基础。包头信息包括: IP 源地址、IP目的地址、封装协议(TCP、UDP、或IP Tunnel)、TCP/UDP源端口、ICMP包类型、包输入接口和包输出接口。如果找到一个匹配，且规则允许这包，这一包则根据路由表中的信息前行。如果找到一个匹配，且规则允许拒绝此包，这一包则被舍弃。如果无匹配规则，一个用户配置的缺省参数将决定此包是前行还是被舍弃。*从属服务的过滤包过滤规则允许Router取舍以一个特殊服务为基

础的信息流，因为大多数服务检测器驻留于众所周知的TCP/UDP端口。例如，Telnet Service 为TCP port 23端口等待远程连接，而SMTP Service为TCP Port 25端口等待输入连接。如要封锁输入Telnet 、SMTP的连接，则Router 舍弃端口值为23,25的所有的数据包。典型的过滤规则有以下几种： .允许特定名单内的内部主机进行Telnet输入对话 .只允许特定名单内的内部主机进行FTP输入对话 .只允许所有Telnet 输出对话 .只允许所有FTP 输出对话 .拒绝来自一些特定外部网络的所有输入信息* 独立于服务的过滤 有些类型的攻击很难用基本包头信息加以鉴别，因为这些独立于服务。一些Router可以用来防止这类攻击，但过滤规则需要增加一些信息，而这些信息只有通过以下方式才能获

CheckPoint防火墙安装手册

防火墙安装操作手册By Shixiong Chen

一、准备安装介质和服务器 安装CheckPoint防火墙基于开放的服务器平台需要准备两点: 如果选用的是Checkpoint的硬件，UTM-1则不需要考虑这些问题。 第一，准备好服务器，服务器最好选择IBM\HP\或者其他大品牌厂商的硬件平台，并且事先确认该品牌和型号的服务器与CheckPoint软件兼容性，将网卡扩展至与真实火墙一致，服务器需要自带光驱。 第二，准备好CheckPoint防火墙安装介质，注意软件的版本号与真实环境火墙一致，同时准备好最新的防火墙补丁。 二、SecurePlatform系统安装过程 硬件服务器初始状态是裸机，将服务器加电后，设置BIOS从光盘启动，将CheckPoint软件介质放入光驱，然后出现如下界面: 敲回车键盘开始安装。出现如下界面，选择OK,跳过硬件检测。

选择安装的操作系统类型，根据自己防火墙操作系统的版本选择。SecurePlatform Pro是带有高级路由和支持Radius的系统版本。 选择键盘支持的语言，默认选择US，按TAB键，继续安装。 配置网络接口，初始我们配置外网接口即可，选择eth0配置IP, 输入IP地址、子网掩码、以及默认网关，然后选择OK,继续。 选择OK.开始格式化磁盘。

格式化完成后开始拷贝文件，最后提示安装完成，按照提示点击OK.系统会自动重新启动。然后出现登陆界面，如下所示。 第一次登陆系统，默认账号和密码都是admin，登陆后需要出入新的密码和管理员账号。 三、CheckPoint防火墙软件安装过程

运行sysconfig 命令，启动安装防火墙包。选择n,继续安装。 打开网络配置页面，选择1,配置主机名，选择3配置DNS服务器，选择4配置网络，选择5配置路由，注意要与生产线设备的配置保持一致，特别是路由要填写完整，主机名、接口IP和子网掩码要填写正确。 配置完成后，选择n,下一步继续配置，如下图所示，选择1配置时区(选择5，9，1)，选择2配置日期，选择3配置本地系统时间，选择4查看配置情况。注意配置时间和日期的格式。完成后选择n， 然后会出现提示是否从tftp服务器下载安装软件，选择n.然后出现如下界面，选择N,继续安装。 选择Y,接受安装许可协议。

防火墙基础知识

(一) 防火墙概念 防火墙不只是一种路由器、主系统或一批向网络提供安全性的系统。相反，防火墙是 一种获取安全性的方法；它有助于实施一个比较广泛的安全性政策，用以确定允许提供的服务和访问。就网络配置、一个或多个主系统和路由器以及其他安全性措施(如代替 静态口令的先进验证)来说，防火墙是该政策的具体实施。防火墙系统的主要用途就是 控制对受保护的网络(即网点)的往返访问。它实施网络访问政策的方法就是逼使各连接 点通过能得到检查和评估的防火墙。 ____________ ___________ | | | | | Computer | | Computer | |__________| |___________| ____________ ________ | | |应| |Packet | ______|________________|__________| 用|_____|Filter |___Internet | 网| |Router | 网点系统| 关| |________| |__________| 路由器和应用网关防火墙范例 防火墙系统可以是路由器，也可以是个人主机、主系统和一批主系统，专门把网 络或子网同那些可能被子网外的主系统滥用的协议和服务隔绝。防火墙系统通常位于等级较高的网关如网点与Internet的连接处，但是防火墙系统可以位于等级较低的网关, 以便为某些数量较少的主系统或子网提供保护。 防火墙基本上是一个独立的进程或一组紧密结合的进程，运行于Router or Server 来控制经过防火墙的网络应用程序的通信流量。一般来说，防火墙置于公共网络(如Inter- -net)人口处。它可以看作是交通警察。它的作用是确保一个单位内的网络与Internet之间所有的通信均符合该单位的安全方针。这些系统基本上是基于TCP/IP,并与实现方法有关，它能实施安全路障并为管理人员提供对下列问题的答案： * 谁在使用网络? * 他们在网上做什么? * 他们什么时间使用过网络?

临界状态滑坡土层参数反演在工程中的应用

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