scholarly journals Chromosomal replication origins in Candida albicans are genetically defined irrespective of their chromosomal context

2020 ◽  
Author(s):  
Sreyoshi Mitra
Keyword(s):  
Candida Albicans ◽  
Dna Replication ◽  
The Novel ◽  
Replication Origins ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Replication Complex ◽  
Intergenic Regions ◽  
Novel Strategy

AbstractInitiation of DNA replication occurs at specialized regions along chromosomes called origins. The knowledge of replication origins is imperative to understand regulation of DNA replication. The properties of replication origins in the pathogenic budding yeast Candida albicans remain enigmatic in the absence of the knowledge of authentic chromosomal origins. Earlier, we identified centromere proximal (pCEN) chromosomal origins on chromosomes 5 and 7 of C. albicans. Here, we identify another centromere-distal (dCEN) chromosomal origin by two-dimensional agarose gel electrophoresis corresponding to a previously reported autonomously replicating sequence (ARS), CARS2. We show that all the identified chromosomal origins are bound by C. albicans homologs of conserved pre-replication complex proteins Orc2 and Mcm2. Previous reports from our lab and others have shown that there is a strong inter-relationship between centromere function and pericentric origin activity, suggesting that these origins are epigenetically regulated. However, we find that short intergenic regions corresponding to each of these origins functions as an ARS element in circular plasmids. Further, we use the novel strategy of in vivo gap repair to demonstrate that circular ARS plasmids can exist independently in vivo in C. albicans. Taken together, these results show that DNA sequence underlies the function of both centromere proximal and centromere distal chromosomal origins in C. albicans.

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Chromosomal DNA replication initiates at the same origins in meiosis and mitosis

1994 ◽  
Vol 14 (5) ◽  
pp. 3524-3534
Author(s):  
I Collins ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Chromosome Iii

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.

scholarly journals Chromosomal DNA replication initiates at the same origins in meiosis and mitosis.

1994 ◽  
Vol 14 (5) ◽  
pp. 3524-3534 ◽  
Author(s):  
I Collins ◽  
C S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Chromosome Iii

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.

scholarly journals The Human Stress-Activated Protein kin17 Belongs to the Multiprotein DNA Replication Complex and Associates In Vivo with Mammalian Replication Origins

2005 ◽  
Vol 25 (9) ◽  
pp. 3814-3830 ◽  
Author(s):  
Laurent Miccoli ◽  
Isabelle Frouin ◽  
Olivia Novac ◽  
Domenic Di Paola ◽  
Francis Harper ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Dna Fragments ◽  
Replication Origins ◽  
Proliferating Cells ◽  
Direct Role ◽  
Replication Complex ◽  
Human Stress

ABSTRACT The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G1/S border and throughout the S phase and was negligible in both G0 and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase α. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an ∼600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

scholarly journals A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 3261-3271 ◽  
Author(s):  
A M Merchant ◽  
Y Kawasaki ◽  
Y Chen ◽  
M Lei ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Replication Origins ◽  
Minichromosome Maintenance ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Dna Replication Initiation ◽  
Initiation Factors

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

scholarly journals The Novel Arylamidine T-2307 MaintainsIn VitroandIn VivoActivity against Echinocandin-Resistant Candida albicans

2014 ◽  
Vol 59 (2) ◽  
pp. 1341-1343 ◽  
Author(s):  
Nathan P. Wiederhold ◽  
Laura K. Najvar ◽  
Annette W. Fothergill ◽  
Rosie Bocanegra ◽  
Marcos Olivo ◽  
...  
Keyword(s):  
Body Weight ◽  
Candida Albicans ◽  
Invasive Candidiasis ◽  
Placebo Control ◽  
The Novel ◽  
Content Type ◽  
Fungal Burden ◽  
Potential Use

ABSTRACTWe evaluated thein vitroandin vivoactivities of the investigational arylamidine T-2307 against echinocandin-resistantCandida albicans. T-2307 demonstrated potentin vitroactivity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistantC. albicansinfections.

A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus

1994 ◽  
Vol 14 (9) ◽  
pp. 5628-5635
Author(s):  
E S Tasheva ◽  
D J Roufa
Keyword(s):  
Dna Replication ◽  
Chinese Hamster ◽  
Housekeeping Gene ◽  
Single Copy ◽  
The Other ◽  
Replication Origins ◽  
Chromosomal Replication ◽  
Dna Binding Sites ◽  
Sequence Encoding ◽  
Copy Dna

Two complementary experimental approaches have been used to identify a chromosomal origin of bidirectional DNA replication within or immediately downstream of the Chinese hamster ribosomal protein S14 gene (RPS14). The replication origin, designated oriS14, maps within a 1.6- to 2.0-kbp region of RPS14 that includes the gene's third and fourth introns, exons IV plus V, and approximately 500 bp of proximal downstream flanking DNA. The nucleic acid sequence encoding oriS14 closely resembles the other mammalian chromosomal replication origins whose primary structures are known. It contains DNA binding sites for a large number of transcription factors, replication proteins, and mammalian oncogenes as well as several dinucleotide repeat motifs, an AT-rich region, and a sequence that is likely to bend the DNA. In contrast to the other well-characterized mammalian replication origins, which are autosomal and therefore carried as two copies per somatic cell, oriS14 is encoded by single-copy DNA within a hemizygous segment of chromosome 2q in CHO-K1 cells. Also, other known mammalian replication origins are situated in nontranscribed, intergenic DNA, whereas the DNA sequence encoding oriS14 substantially overlaps the transcribed portion of a constitutively expressed housekeeping gene.

scholarly journals A Coordinated Temporal Interplay of Nucleosome Reorganization Factor, Sister Chromatin Cohesion Factor, and DNA Polymerase α Facilitates DNA Replication

2004 ◽  
Vol 24 (21) ◽  
pp. 9568-9579 ◽  
Author(s):  
Yanjiao Zhou ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
S Phase ◽  
Terminal Region ◽  
Replication Complex ◽  
Dna Polymerase Α ◽  
Chromatid Cohesion ◽  
Phase Progression

ABSTRACT DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase α (Polα). The catalytic subunit of budding yeast Polα (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polα (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polα protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

scholarly journals Origin Replication Complex Binding, Nucleosome Depletion Patterns, and a Primary Sequence Motif Can Predict Origins of Replication in a Genome with Epigenetic Centromeres

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Hung-Ji Tsai ◽  
Joshua A. Baller ◽  
Ivan Liachko ◽  
Amnon Koren ◽  
Laura S. Burrack ◽  
...  
Keyword(s):  
Machine Learning ◽  
Candida Albicans ◽  
Dna Replication ◽  
Content Type ◽  
Replication Complex ◽  
Origins Of Replication ◽  
A Genome ◽  
Bona Fide ◽  
Origin Replication Complex ◽  
Origin Function

ABSTRACTOrigins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeastCandida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns inSaccharomyces cerevisiaeandKluyveromyces lactisto train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in theC. albicansgenome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A “mini-ARS screen” identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion.IMPORTANCEDNA replication machinery is highly conserved, yet the definition of exactly what specifies a replication origin differs in different species. Here, we utilized computational genomics to predict origin locations inCandida albicansby combining locations of binding sites for the conserved origin replication complex, necessary for replication initiation, together with chromatin organization patterns. We identified predicted sequences that exhibited bona fide origin function and developed a linear plasmid assay to delimit the DNA fragments necessary for origin function. Additionally, we found that a short AC-rich motif, which is enriched in predicted origins, is required for origin function. Thus, we demonstrated a new machine learning paradigm for identification of potential origins from a genome with no prior information. Furthermore, this work suggests thatC. albicanshas two different types of origins: “hard-wired” arm origins that rely upon specific sequence motifs and “epigenetic” centromeric origins that are recruited to kinetochores in a sequence-independent manner.

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