Visualization of replication initiation and elongation in Drosophila

Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2–7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre–replication complex component, double-parked protein/cell division cycle 10–dependent transcript 1, is not only necessary for proper MCM2–7 localization, but, unexpectedly, is present during elongation.

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Titration of Four Replication Factors Is Essential for the Xenopus laevis Midblastula Transition

Science ◽

10.1126/science.1241530 ◽

2013 ◽

Vol 341 (6148) ◽

pp. 893-896 ◽

Cited By ~ 129

Author(s):

Clara Collart ◽

George E. Allen ◽

Charles R. Bradshaw ◽

James C. Smith ◽

Philip Zegerman

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Xenopus Laevis ◽

Replication Initiation ◽

Midblastula Transition ◽

Multiple Events ◽

Zygotic Transcription ◽

Replication Factors

The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors—Cut5, RecQ4, Treslin, and Drf1—are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.

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Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins

Journal of Virology ◽

10.1128/jvi.01968-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1458-1464 ◽

Cited By ~ 37

Author(s):

Kevin Nash ◽

Weijun Chen ◽

Nicholas Muzyczka

Keyword(s):

Dna Replication ◽

Productive Infection ◽

Nuclear Antigen ◽

Minichromosome Maintenance ◽

Adeno Associated Virus ◽

Rep Protein ◽

Mcm Complex ◽

Factor C

ABSTRACT Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase δ as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase δ, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase δ inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase δ play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.

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Regulation of DNA Replication Licensing and Re-Replication by Cdt1

International Journal of Molecular Sciences ◽

10.3390/ijms22105195 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5195

Author(s):

Hui Zhang

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Genome Stability ◽

E3 Ligase ◽

S Phase ◽

Replication Initiation ◽

Nuclear Antigen ◽

Repair Synthesis ◽

Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

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Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0426 ◽

2007 ◽

Vol 18 (1) ◽

pp. 129-141 ◽

Cited By ~ 53

Author(s):

Yasunari Takami ◽

Tatsuya Ono ◽

Tatsuo Fukagawa ◽

Kei-ichi Shibahara ◽

Tatsuo Nakayama

Keyword(s):

Dna Replication ◽

Essential Role ◽

Chromatin Assembly ◽

Nuclear Antigen ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

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Regulation of Replication Licensing by Acetyltransferase Hbo1

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.1098-1108.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 1098-1108 ◽

Cited By ~ 132

Author(s):

Masayoshi Iizuka ◽

Tomoko Matsui ◽

Haruhiko Takisawa ◽

M. Mitchell Smith

Keyword(s):

Dna Replication ◽

Origin Recognition Complex ◽

Cyclin Dependent Kinase ◽

Chromatin Binding ◽

Regulatory Factor ◽

Minichromosome Maintenance ◽

Egg Extracts ◽

Sequential Binding ◽

Initiation Of Dna Replication ◽

Prereplicative Complex

ABSTRACT The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.

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Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms

10.1101/2020.07.04.187286 ◽

2020 ◽

Cited By ~ 3

Author(s):

Y. Hu ◽

A. Tareen ◽

Y-J. Sheu ◽

W. T. Ireland ◽

C. Speck ◽

...

Keyword(s):

Dna Replication ◽

Gene Silencing ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Separate Analysis ◽

Genome Replication ◽

Sequence Specificity ◽

Sequence Motifs ◽

Origin Firing ◽

Α Helix

AbstractDNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

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Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5767-5777.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5767-5777 ◽

Cited By ~ 54

Author(s):

Amit Vas ◽

Winnie Mok ◽

Janet Leatherwood

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Origin Recognition Complex ◽

Safety Net ◽

Replication Initiation ◽

Initiation Factor ◽

Phosphorylation Sites ◽

Cdc2 Kinase ◽

Origin Recognition ◽

Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

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Essential role of human CDT1 in DNA replication and chromatin licensing

Journal of Cell Science ◽

10.1242/jcs.115.7.1435 ◽

2002 ◽

Vol 115 (7) ◽

pp. 1435-1440 ◽

Cited By ~ 2

Author(s):

Mickael Rialland ◽

Francesco Sola ◽

Corrado Santocanale

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Complex Formation ◽

Fission Yeast ◽

Origin Recognition Complex ◽

Essential Role ◽

Large Body ◽

Transformed Cells ◽

Minichromosome Maintenance

Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication. A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA. In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein. We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells. We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin. Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle.

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Fibroblast proliferation during myocardial development in rats is regulated by IGF-1 receptors

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.269.3.h943 ◽

1995 ◽

Vol 269 (3) ◽

pp. H943-H951 ◽

Cited By ~ 3

Author(s):

K. Reiss ◽

W. Cheng ◽

J. Kajstura ◽

E. H. Sonnenblick ◽

L. G. Meggs ◽

...

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Cardiac Fibroblasts ◽

Nuclear Antigen ◽

Dna Polymerase Alpha ◽

Myocardial Development ◽

Left And Right ◽

Proliferating Cell

To determine whether the growth of cardiac fibroblasts during development is modulated by the insulin-like growth factor (IGF)-1 receptor (IGF-1R), the expression of IGF-1, IGF-2, and IGF-1R was determined in fibroblasts from fetal and postnatal hearts. The expression of proliferating cell nuclear antigen (PCNA) and DNA polymerase-alpha was also evaluated in combination with the estimation of DNA replication. In comparison with fetal hearts, at postnatal day 21, fibroblast expression of IGF-1R mRNA, IGF-2, PCNA, and DNA polymerase-alpha was reduced by 77, 70, 80, and 86%, respectively. Moreover, IGF-1R protein decreased by 48% at 21 days. Bromodeoxyuridine labeling decreased by 88 and 89% in the left and right ventricle, respectively, at this time. Two different antisense oligodeoxynucleotides to IGF-1R reduced DNA replication by 60 and 44% in fibroblasts in culture. In addition, this intervention markedly attenuated the growth response of fibroblasts to IGF-1 or serum. In conclusion, the IGF-1R system appears to play a major role in the regulation of fibroblast growth in the heart in vivo.

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Dynamics of DNA replication in a eukaryotic cell

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818680116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4973-4982 ◽

Cited By ~ 18

Author(s):

Thomas Kelly ◽

A. John Callegari

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Origin Recognition Complex ◽

Complex Dynamics ◽

Replication Timing ◽

Eukaryotic Cell ◽

Replication Initiation ◽

Integrative Model ◽

Genomic Locus ◽

Two Factors

Each genomic locus in a eukaryotic cell has a distinct average time of replication during S phase that depends on the spatial and temporal pattern of replication initiation events. Replication timing can affect genomic integrity because late replication is associated with an increased mutation rate. For most eukaryotes, the features of the genome that specify the location and timing of initiation events are unknown. To investigate these features for the fission yeast, Schizosaccharomyces pombe, we developed an integrative model to analyze large single-molecule and global genomic datasets. The model provides an accurate description of the complex dynamics of S. pombe DNA replication at high resolution. We present evidence that there are many more potential initiation sites in the S. pombe genome than previously identified and that the distribution of these sites is primarily determined by two factors: the sequence preferences of the origin recognition complex (ORC), and the interference of transcription with the assembly or stability of prereplication complexes (pre-RCs). We suggest that in addition to directly interfering with initiation, transcription has driven the evolution of the binding properties of ORC in S. pombe and other eukaryotic species to target pre-RC assembly to regions of the genome that are less likely to be transcribed.

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