In Vivo Association of Ku with Mammalian Origins of DNA Replication

Novac, Olivia; Matheos, Diamanto; Araujo, Felipe D.; Price, Gerald B.; Zannis‐Hadjopoulos, Maria

doi:10.1091/mbc.12.11.3386

Cited by 54 publications

(85 citation statements)

References 90 publications

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“…Taken together with previous observations, our results suggest that Ku is endowed with two modes of binding to silent regions of DNA: it can bind telomeric regions directly via its DNA end-binding activity and it can bind HML and HMR as a result of protein-protein interactions. Previous observations have also implicated Ku in binding to internal chromosomal loci and suggest that Ku may play a role in the activation of transcription and possibly in initiation of replication (Barnes and Rio 1997;Ruiz et al 1999;Novac et al 2001;Walker et al 2001;Schild-Poulter et al 2003;Sibani et al 2005;Grote et al 2006;Shi et al 2007;Rampakakis et al 2008). The data presented here support and extend the data indicating that Ku binds to internal chromosomal loci and broaden our knowledge of the number of processes in which Ku plays a role at internal loci to include silencing.…”

Section: Discussionsupporting

confidence: 85%

The DNA End-Binding Protein Ku Regulates Silencing at the InternalHMLandHMRLoci inSaccharomyces cerevisiae

2008

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Heterochromatin resides near yeast telomeres and at the cryptic mating-type loci, HML and HMR, where it silences transcription of the a-and a-mating-type genes, respectively. Ku is a conserved DNA end-binding protein that binds telomeres and regulates silencing in yeast. The role of Ku in silencing is thought to be limited to telomeric silencing. Here, we tested whether Ku contributes to silencing at HML or HMR . Mutant analysis revealed that yKu70 and Sir1 act collectively to silence the mating-type genes at HML and HMR. In addition, loss of yKu70 function leads to expression of different reporter genes inserted at HMR. Quantitative chromatin-immunoprecipitation experiments revealed that yKu70 binds to HML and HMR and that binding of Ku to these internal loci is dependent on Sir4. The interaction between yKu70 and Sir4 was characterized further and found to be dependent on Sir2 but not on Sir1, Sir3, or yKu80. These observations reveal that, in addition to its ability to bind telomeric DNA ends and aid in the silencing of genes at telomeres, Ku binds to internal silent loci via protein-protein interactions and contributes to the efficient silencing of these loci.

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Section: Discussionsupporting

confidence: 85%

The DNA End-Binding Protein Ku Regulates Silencing at the InternalHMLandHMRLoci inSaccharomyces cerevisiae

2008

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“…Instead, Ku and Sir4 may be relying on some other silencer-recognition mechanism. In this regard, it is intriguing that the effects of yeast Ku on ORC binding in vitro have been reported (Shakibai et al 1996) and that in mammalian cells Ku associates with replication origins and has been found in a complex with known replication proteins, including ORC2 (Novac et al 2001;Matheos et al 2002Matheos et al , 2003. In addition, recent two-hybrid analyses of Orc subunits report an interaction between Orc2 and Sir4 (Matsuda et al 2007).…”

Section: Discussionmentioning

confidence: 99%

The Ku Complex in Silencing the Cryptic Mating-Type Loci ofSaccharomyces cerevisiae

Patterson

Fox

2008

Genetics

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Sir1 establishes transcriptional silencing at the cryptic mating-type loci HMR and HML (HM loci) by recruiting the three other Sir proteins, Sir2, -3, and -4, that function directly in silenced chromatin. However, SIR1-independent mechanisms also contribute to recruiting the Sir2-4 proteins to the HM loci. A screen to elucidate SIR1-independent mechanisms that establish HMR silencing identified a mutation in YKU80. The role for Ku in silencing both HMR and HML was masked by SIR1. Ku's role in silencing the HM loci was distinct from its shared role with the nuclear architecture protein Esc1 in tethering the HM loci and telomeres to the nuclear periphery. The ability of high-copy SIR4 to rescue HMR silencing defects in sir1D cells required Ku, and chromatin immunoprecipitation (ChIP) experiments provided evidence that Ku contributed to Sir4's physical association with the HM loci in vivo. Additional ChIP experiments provided evidence that Ku functioned directly at the HM loci. Thus Ku and Sir1 had overlapping roles in silencing the HM loci.T RANSCRIPTIONAL repression of the cryptic matingtype loci HMR and HML (the HM loci) in the yeast Saccharomyces cerevisiae occurs through a mechanism called transcriptional silencing. Silencing describes the formation of a specialized ''silent'' chromatin structure that is analogous on many levels to metazoan heterochromatin (reviewed in Rusche et al. 2003). Specifically, silencing causes heritable, position-dependent, long-range transcriptional repression that relies on interactions between nucleosomes, chromatin-modifying enzymes, and nonhistone chromatin-binding proteins. In addition, several proteins and chromatin modifications involved in silencing are conserved in metazoans and have roles in heterochromatin (reviewed in Perrod and Gasser 2003). An understanding of silencing requires identification of the proteins and protein-protein and protein-DNA interactions required to target silent chromatin formation to the appropriate domains in the genome. A challenge to obtaining a comprehensive description of silencing at the HM loci using genetic approaches is that several overlapping mechanisms contribute to a robustness in silent chromatin structure such that the functions of key proteins and molecular interactions can be masked. In this study we analyzed silencing compromised by a deletion of SIR1 (silent information regulator; sir1D) and discovered that the yeast Ku complex had a redundant role with Sir1 in silencing the HM loci.

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“…The efficiency of this approach has been demonstrated in a number of studies (47,52,58,(61)(62)(63)(64)(65)(66)(67)(68)(69)(70). Formaldehyde treatment of cells readily produces protein-protein and protein-DNA cross-linked complexes.…”

Section: Discussionmentioning

confidence: 99%

The Human Cruciform-binding Protein, CBP, Is Involved in DNA Replication and Associates in Vivo with Mammalian Replication Origins

Novac

Álvarez

Pearson

et al. 2002

Journal of Biological Chemistry

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We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3␤, -⑀, -␥, andisoforms with these origins was found to be ϳ9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3␤, -⑀, -␥, and -isoforms with ors8 and ors12 was found at the G 1 /S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G 1 /S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3␤, -⑀, -␥, and -antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3⑀, -␥, and -antibodies alone or in combination inhibited p186 replication by ϳ50 -80%, while anti-14-3-3␤ antibodies had a lesser effect (ϳ25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.Inverted repeat sequences (IRs) 1 are a common feature of prokaryotic and eukaryotic regulatory regions, and their distribution is nonrandom and clustered (1). Promoters (2, 3), terminators (4), amplified genes (5), and origins of DNA replication from prokaryotes (6 -8), viruses (9), and eukaryotes (10) We and others have previously demonstrated the involvement of cruciform structures in the initiation of mammalian DNA replication (20, 23-25) (reviewed in Ref. 16) We previously identified and isolated from human cells (HeLa) a cruciform-specific binding protein, CBP (26), of 66 kDa with binding specificity for cruciform-containing DNA (26). CBP binds at the base of four-way junctions (27), interacting with them in a different manner from other proteins known to bind such junctions (26,27).CBP is a member of the 14-3-3 protein family, a highly conserved family (28) of proteins through plants, invertebrates, and higher eukaryotes (reviewed in Ref. 29). 14-3-3 proteins are multifunctional, involved in diverse cellular processes, such as neurotransmitter biosynthesis, signal transduction pathways, and cell cycle control (reviewed in Ref. 30). 14-3-3-associated proteins include rec...

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In Vivo Association of Ku with Mammalian Origins of DNA Replication

Cited by 54 publications

References 90 publications

The DNA End-Binding Protein Ku Regulates Silencing at the InternalHMLandHMRLoci inSaccharomyces cerevisiae

The DNA End-Binding Protein Ku Regulates Silencing at the InternalHMLandHMRLoci inSaccharomyces cerevisiae

The Ku Complex in Silencing the Cryptic Mating-Type Loci ofSaccharomyces cerevisiae

The Human Cruciform-binding Protein, CBP, Is Involved in DNA Replication and Associates in Vivo with Mammalian Replication Origins

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