Recent Replication Papers

Malone RE, Haring SJ, Foreman KE, Pansegrau ML, Smith SM, Houdek DR, Carpp L, Shah B, Lee KE

Eukaryot Cell 3:598-609
Jun , 2004

Two of the unique events that occur in meiosis are high levels of genetic recombination and the reductional division. Our previous work demonstrated that the REC102, REC104, REC114, and RAD50 genes, required to initiate meiotic recombination in Saccharomyces cerevisiae, are needed for the proper timing of the first meiotic (MI) division. If these genes are absent, the MI division actually begins at an earlier time. This paper demonstrates that the meiotic recombination genes MER2/REC107, SPO11, and MRE2 and the synaptonemal complex genes HOP1 and RED1 are also required for the normal delay of the MI division. A rec103/ski8 mutant starts the MI division at the same time as in wild-type cells. Our data indicate no obvious correlation between the timing of premeiotic S phase and the timing of the first division in Rec(-) mutants. Cells with rec102 or rec104 mutations form MI spindles before wild-type cells, suggesting that the initiation signal acts prior to spindle formation. Neither RAD9 nor RAD24 is needed to transduce the signal, which delays the first division. The timing of the MI division in RAD24 mutants indicates that the pachytene checkpoint is not active in Rec(+) cells and suggests that the coordination between recombination and the MI division in wild-type cells may occur primarily due to the initiation signal. Finally, at least one of the targets of the recombination initiation signal is the NDT80 gene, a transcriptional regulator of middle meiotic gene expression required for the first division.

Kinoshita Y, Johnson EM

J Biol Chem HASH(0x8b7960):HASH(0x8b8894)
Jun 9, 2004

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6 and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G1 and early S on a 198 bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G1. MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G2, reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G2, at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G1 followed by a recruitment to multiple sites in chromatin subsequent to replication.

Blander G, Guarente L

Annu Rev Biochem 73:417-35
, 2004

The yeast SIR protein complex has been implicated in transcription silencing and suppression of recombination. The Sir complex represses transcription at telomeres, mating-type loci, and ribosomal DNA. Unlike SIR3 and SIR4, the SIR2 gene is highly conserved in organisms ranging from archaea to humans. Interestingly, Sir2 is active as an NAD+-dependent deacetylase, which is broadly conserved from bacteria to higher eukaryotes. In this review, we discuss the role of NAD+, the unusual products of the deacetylation reaction, the Sir2 structure, and the Sir2 chemical inhibitors and activators that were recently identified. We summarize the current knowledge of the Sir2 homologs from different organisms, and finally we discuss the role of Sir2 in caloric restriction and aging.

Li CJ, Vassilev A, DePamphilis ML

Mol Cell Biol 24:5875-86
Jul , 2004

The eukaryotic origin recognition complex (ORC) selects the genomic sites where prereplication complexes are assembled and DNA replication begins. In proliferating mammalian cells, ORC activity appears to be regulated by reducing the affinity of the Orc1 subunit for chromatin during S phase and then preventing reformation of a stable ORC-chromatin complex until mitosis is completed and a nuclear membrane is assembled. Here we show that part of the mechanism by which this is accomplished is the selective association of Orc1 with Cdk1 (Cdc2)/cyclin A during the G(2)/M phase of cell division. This association accounted for the appearance in M-phase cells of hyperphosphorylated Orc1 that was subsequently dephosphorylated during the M-to-G(1) transition. Moreover, inhibition of Cdk activity in metaphase cells resulted in rapid binding of Orc1 to chromatin. However, chromatin binding was not mediated through increased affinity of Orc1 for Orc2, suggesting that additional events are involved in the assembly of functional ORC-chromatin sites. These results reveal that the same cyclin-dependent protein kinase that initiates mitosis in mammalian cells also concomitantly inhibits assembly of functional ORC-chromatin sites.

Ekholm-Reed S, Méndez J, Tedesco D, Zetterberg A, Stillman B, Reed SI

J Cell Biol HASH(0x8bbdb4):HASH(0x8bd0f8)
Jun 14, 2004

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2-7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E-deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E-mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.

Li W, Kim SM, Lee J, Dunphy WG

J Cell Biol HASH(0x8bbbf8):HASH(0x8bcfb4)
Jun 14, 2004

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIalpha (Xtop3alpha) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3alpha has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.

Yoshida K, Inoue I

Oncogene HASH(0x8bba60):HASH(0x8bbd18)
Jun 14, 2004

MCM10 and TopBP1 function in the initiation of DNA replication, by regulating the chromatin binding of the DNA polymerase alpha loading factor, CDC45. TopBP1 is also known as a DNA damage response protein. In this study, we showed that the transcription of human MCM10 and TopBP1 is activated by transcription factors E2F1-3, but not by factors E2F4-7. Analysis of various MCM10 and TopBP1 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the E2F1-induced activation of MCM10 and TopBP1 gene transcription, which is further suppressed by pRb. The promoter activities of human MCM10 and TopBP1 were demonstrated to be growth dependent via the E2F-responsive sequence. Although E2F1 was stabilized by ultraviolet (UV) irradiation, the mRNA expression level of TopBP1 was suppressed in HCT116 human diploid colon cancer cells. We showed, by performing chromatin immunoprecipitation that, in response to UV irradiation but not doxorubicin treatment, E2F4 accumulated on the MCM10 and TopBP1 promoters. Our data suggest a model in which UV irradiation-induced DNA damage depends, at least in part, on the accumulation of the E2F4 transcription factor on the MCM10 and TopBP1 promoters, which results in suppression of DNA replication.Oncogene advance online publication, 14 June 2004; doi:10.1038/sj.onc.1207829

Dodson GE, Shi Y, Tibbetts RS

J Biol Chem HASH(0x6b784c):HASH(0x8bcdec)
Jun 14, 2004

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)-binding complex comprised of 70 kDa (RPA1), 32 kDa (RPA2), and 14 kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR which, in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. However, the requirement of RPA for ATR recruitment and activation is controversial and the cellular consequences of RPA functional deficiency have not been investigated. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused S phase slowing, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the HU- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB. These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways in response to genotoxic stress.

Yamada Y, Nakagawa T, Masukata H

Mol Biol Cell HASH(0x77cd44):HASH(0x8bbd9c)
Jun 11, 2004

Monitoring Editor: Keith Yamamoto Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of pre-initiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably due to a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.

Chastain PD, Bowers JL, Lee DG, Bell SP, Griffith JD

J Biol Chem HASH(0x773be8):HASH(0x774b6c)
Jun 16, 2004

The Saccharomyces cerevisiae origin recognition complex (ORC) is comprised of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multi-protein complex by electron microscopy each subunit was tagged on either its C- or N-terminus with biotin and assembled into a complex with the 5 other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N- and C-termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.

Weisshart K, Pestryakov P, Smith RW, Hartmann H, Kremmer E, Lavrik O, Nasheuer HP

J Biol Chem HASH(0x8ba8f0):HASH(0x8bb824)
Jun 17, 2004

The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPADp14, consisting of p70 and p32. RPADp14 was able to bind single-stranded DNA (ssDNA) but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADp14 complex p32 only minimally recognized the 3-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPADp14 efficiently supported bi-directional unwinding of double-stranded DNA and interacted with both the Simian Virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on ssDNA requires all three subunits to support DNA replication.

Sawyer SL, Cheng IH, Chai W, Tye BK

J Mol Biol 340:195-202
Jul 2, 2004

Mcm10 has recently been found to play a crucial role in multiple steps of the DNA replication initiation process in eukaryotes. Here, we have examined the role of Mcm10 in assembling initiation factors at a well-characterized yeast replication origin, ARS1. We find that the pre-replication complex (pre-RC) components Cdc6 and Mcm7 associate with ARS1 in the mcm10-1 mutant, suggesting that establishment of the pre-RC is not compromised in this mutant. Association of Cdc45 with ARS1 is reduced in the mcm10-1 mutant, suggesting that Mcm10 is involved in recruiting Cdc45 to the pre-RC. We find that overexpression of either Mcm10-1 or Cdc45 suppresses the growth defect of mcm10-1, and that a physical interaction between Cdc45 and Mcm10 is disrupted in the mcm10-1 mutant. Our results show that interaction between the Mcm10 and Cdc45 proteins facilitates the recruitment of Cdc45 onto the ARS1 origin.

Miyake T, Reese J, Loch CM, Auble DT, Li R

J Biol Chem HASH(0x771978):HASH(0x77291c)
Jun 10, 2004

ARS-binding factor 1 (Abf1p) is an essential sequence-specific transcription factor in Saccharomyces cerevisiae that participates in multiple nuclear events including DNA replication, transcription activation, and gene silencing. Numerous gene-specific analyses have implicated Abf1p in the transcriptional control of genes involved in a diverse range of cellular functions, leading to the notion that Abf1p acts as a global transcriptional regulator. Here we report findings from a genome-wide comparison of the gene expression profiles in the wild-type and abf1-1 temperature sensitive mutant. The study identifies a total of 86 Abf1p-regulated genes (1.4% of the genome), of which 50 are activated and 36 repressed by Abf1p. Interestingly, Abf1p binds to its own promoter in vivo and strongly represses its own transcription, suggesting a potential negative regulatory loop in Abf1p-mediated gene regulation. Comparison of our microarray data with the available databases reveals a significant overlap of genes regulated by Abf1p and those by several general transcription factors such as Mot1p and TAFs. Different mutant alleles of abf1 affect Abf1p-mediated transcription in a gene-dependent manner. Furthermore, Abf1p in vivo is associated with the promoter region of most Abf1p-activated but not with that of most Abf1p-repressed genes. Taken together, these results strongly suggest distinct underlying mechanisms by which Abf1p regulates gene expression.

Su'etsugu M, Takata M, Kubota T, Matsuda Y, Katayama T

Genes Cells 9:509-22
Jun , 2004

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding.

Majka J, Burgers PM

Prog Nucleic Acid Res Mol Biol 78:227-60
, 2004

The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA(+) protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44?62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

Cortez D, Glick G, Elledge SJ

Proc Natl Acad Sci U S A HASH(0x8a52ec):HASH(0x8a5664)
Jun 21, 2004

The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. We describe four lines of evidence that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex. First, ATM phosphorylates MCM3 on S535 in response to ionizing radiation. Second, ATR phosphorylates MCM2 on S108 in response to multiple forms of DNA damage and stalling of replication forks. Third, ATR-interacting protein (ATRIP)-ATR interacts with MCM7. Fourth, reducing the amount of MCM7 in cells disrupts checkpoint signaling and causes an intra-S-phase checkpoint defect. Thus, the MCM complex is a platform for multiple DNA damage-dependent regulatory signals that control DNA replication.

Norio P, Schildkraut CL

PLoS Biol 2:E152
Jun , 2004

In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.

PLoS Biol 2:E184
Jun , 2004

Miyata T, Oyama T, Mayanagi K, Ishino S, Ishino Y, Morikawa K

Nat Struct Mol Biol HASH(0x8a7b0c):HASH(0x8aa074)
Jun 20, 2004

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC-PCNA-DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.

McPherson JP, Lemmers B, Chahwan R, Pamidi A, Migon E, Matysiak-Zablocki E, Moynahan ME, Essers J, Hanada K, Poonepalli A, Sanchez-Sweatman O, Khokha R, Kanaar R, Jasin M, Hande MP, Hakem R

Science 304:1822-6
Jun 18, 2004

Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus81-/- mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to gamma-irradiation. Remarkably, both homozygous Mus81-/- and heterozygous Mus81+/- mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression.

Danzer JR, Wallrath LL

Development HASH(0x8b839c):HASH(0x8b8714)
Jun 23, 2004

Heterochromatin Protein 1 (HP1) is a structural component of silent chromatin at telomeres and centromeres. Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner. Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes. HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9. The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber. To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E. coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats. Association of lacI-HP1 with the repeats resulted in silencing of both reporter genes and correlated with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin. Chromatin immunoprecipitation experiments demonstrated that HP1 spread bi-directionally from the tethering site and associated with the silenced reporter transgenes. To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated. Silencing was minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner.

De Bruin RA, McDonald WH, Kalashnikova TI, Yates J, Wittenberg C

Cell 117:887-98
Jun 25, 2004

G1-specific transcriptional activation by Cln3/CDK initiates the budding yeast cell cycle. To identify targets of Cln3/CDK, we analyzed the SBF and MBF transcription factor complexes by multidimensional protein interaction technology (MudPIT). Whi5 was identified as a stably bound component of SBF but not MBF. Inactivation of Whi5 leads to premature expression of G1-specific genes and budding, whereas overexpression retards those processes. Whi5 inactivation bypasses the requirement for Cln3 both for transcriptional activation and cell cycle initiation. Whi5 associates with G1-specific promoters via SBF during early G1 phase, then dissociates coincident with transcriptional activation. Dissociation of Whi5 is promoted by Cln3 in vivo. Cln/CDK phosphorylation of Whi5 in vitro promotes its dissociation from SBF complexes. Mutation of putative CDK phosphorylation sites, at least five of which are phosphorylated in vivo, strongly reduces SBF-dependent transcription and delays cell cycle initiation. Like mammalian Rb, Whi5 is a G1-specific transcriptional repressor antagonized by CDK.

Prasanth SG, Prasanth KV, Siddiqui K, Spector DL, Stillman B

EMBO J HASH(0x8b7458):HASH(0x8b845c)
Jun 24, 2004

The initiation of DNA replication in S phase requires the prior assembly of an origin recognition complex (ORC)-dependent pre-replicative complex on chromatin during G1 phase of the cell division cycle. In human cells, the Orc2 subunit localized to the nucleus as expected, but it also localized to centrosomes throughout the entire cell cycle. Furthermore, Orc2 was tightly bound to heterochromatin and heterochromatin protein 1alpha (HP1alpha) and HP1beta in G1 and early S phase, but during late S, G2 and M phases tight chromatin association was restricted to centromeres. Depletion of Orc2 by siRNA caused multiple phenotypes. A population of cells showed an S-phase defect with little proliferating cell nuclear antigen (PCNA) on chromatin, although MCM proteins remained. Orc2 depletion also disrupted HP1 localization, but not histone-H3-lysine-9 methylation at prominent heterochromatic foci. Another subset of Orc2-depleted cells containing replicated DNA arrested with abnormally condensed chromosomes, failed chromosome congression and multiple centrosomes. These results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle.

Bennett RJ, Keck JL

Crit Rev Biochem Mol Biol 39:79-97
, 2004

RecQ family helicases play important roles in coordinating genome maintenance pathways in living cells. In the absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including increased mitotic recombination, elevated chromosome missegregation, hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three of the five human RecQ family members give rise to genetic disorders associated with a predisposition to cancer and premature aging, highlighting the importance of RecQ proteins and their cellular activities for human health. Current evidence suggests that RecQ proteins act at multiple steps in DNA replication, including stabilization of replication forks and removal of DNA recombination intermediates, in order to maintain genome integrity. The cellular basis of RecQ helicase function may be explained through interactions with multiple components of the DNA replication and recombination machinery. This review focuses on biochemical and structural aspects of the RecQ helicases and how these features relate to their known cellular function, specifically in preventing excessive recombination.