scholarly journals Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

2019 ◽  
Author(s):  
Jeziel D. Damasceno ◽  
João Reis-Cunha ◽  
Kathryn Crouch ◽  
Craig Lapsley ◽  
Luiz R. O. Tosi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Dna Synthesis ◽  
Leishmania Major ◽  
Critical Role ◽  
Dna Lesions ◽  
Replication Initiation ◽  
Conditional Knockout ◽  
Genome Replication ◽  
Genome Wide

AbstractHomologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal, but in each case growth slows with time and leads to DNA damage, accumulation of cells with aberrant DNA content, and genome-wide mutation. Despite these similarities, we show that only loss of RAD51 and RAD51-3 impairs DNA synthesis, and that the factors act in distinct ways. Finally, we reveal that loss of RAD51 has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

scholarly journals Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

PLoS Genetics ◽  
2020 ◽  
Vol 16 (7) ◽  
pp. e1008828 ◽  
Author(s):  
Jeziel D. Damasceno ◽  
João Reis-Cunha ◽  
Kathryn Crouch ◽  
Dario Beraldi ◽  
Craig Lapsley ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Leishmania Major ◽  
Critical Role ◽  
Conditional Knockout ◽  
Genome Replication

scholarly journals Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms

2020 ◽  
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.

scholarly journals Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

2020 ◽  
Vol 10 (6) ◽  
pp. 2057-2068 ◽  
Author(s):  
Jessica R. Eisenstatt ◽  
Lars Boeckmann ◽  
Wei-Chun Au ◽  
Valerie Garcia ◽  
Levi Bursch ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Initiation ◽  
Centromeric Chromatin ◽  
Dna Replication Initiation ◽  
Link Type ◽  
Genome Wide ◽  
A Genome ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.

scholarly journals The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication

2020 ◽  
Vol 44 (3) ◽  
pp. 351-368 ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Christophe Possoz ◽  
David R F Leach
Keyword(s):  
Dna Replication ◽  
Cell Viability ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Initiation ◽  
Genome Replication ◽  
Chromosomal Dna ◽  
Dsb Repair ◽  
Bacterial Dna ◽  
Dna Double Strand Break

ABSTRACT It is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.

scholarly journals Genome-wide analysis of DNA turnover and gene expression in stationary-phase Saccharomyces cerevisiae

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1758-1771 ◽  
Author(s):  
A. de Morgan ◽  
L. Brodsky ◽  
Y. Ronin ◽  
E. Nevo ◽  
A. Korol ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Stationary Phase ◽  
Dna Synthesis ◽  
Exponential Phase ◽  
Yeast Cells ◽  
Genome Wide ◽  
Dna Turnover ◽  
Genomic Scale ◽  
Dna Maintenance

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2′-deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdU-labelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes.

The programme of DNA replication: beyond genome duplication

2013 ◽  
Vol 41 (6) ◽  
pp. 1720-1725 ◽  
Author(s):  
Blanca Gómez-Escoda ◽  
Pei-Yun Jenny Wu
Keyword(s):  
Dna Replication ◽  
Cell Growth ◽  
Dna Synthesis ◽  
Genetic Information ◽  
Replication Origin ◽  
Genome Duplication ◽  
Replication Initiation ◽  
Origin Activation ◽  
Key Steps ◽  
Cell Growth And Proliferation

The accurate duplication and transmission of genetic information is critical for cell growth and proliferation, and this is ensured in part by the multi-layered regulation of DNA synthesis. One of the key steps in this process is the selection and activation of the sites of replication initiation, or origins, across the genome. Interestingly, origin usage changes during development and in different pathologies, suggesting an integral interplay between the establishment of replication initiation along the chromosomes and cellular function. The present review discusses how the spatiotemporal organization of replication origin activation may play crucial roles in the control of biological events.

scholarly journals Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38

2007 ◽  
Vol 189 (6) ◽  
pp. 2392-2400 ◽  
Author(s):  
Kazuteru Aoki ◽  
Miki Shinohara ◽  
Tateo Itoh
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Replication Initiation ◽  
Mobility Shift ◽  
Rep Protein ◽  
Rep Proteins ◽  
Mobility Shift Assay ◽  
Hand Region ◽  
A Site ◽  
Specificity Determinants

ABSTRACT The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, α and β, in the origins as specificity determinants and showed that when each component of the region A-site α pair and the region B-site β pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site α. By using an electrophoretic mobility shift assay, we demonstrated that region B and site β play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site α were involved in the efficient unwinding of several nucleotide residues around site α, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.

scholarly journals The chromatin remodeler ALC1 underlies resistance to PARP inhibitor treatment

2020 ◽  
Vol 6 (51) ◽  
pp. eabb8626
Author(s):  
Szilvia Juhász ◽  
Rebecca Smith ◽  
Tamás Schauer ◽  
Dóra Spekhardt ◽  
Hasan Mamar ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Parp Inhibitor ◽  
Parp Inhibitors ◽  
Dna Lesions ◽  
End Joining ◽  
Multiple Tumor ◽  
Genome Wide ◽  
A Genome ◽  
Inhibitor Resistance ◽  
Tumor Types

Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the treatment of BRCA-deficient cancers, with treatments currently extending toward other homologous recombination defective tumors. In a genome-wide CRISPR knockout screen with olaparib, we identify ALC1 (Amplified in Liver Cancer 1)—a cancer-relevant poly(ADP-ribose)-regulated chromatin remodeling enzyme—as a key modulator of sensitivity to PARP inhibitor. We found that ALC1 can remove inactive PARP1 indirectly through binding to PARylated chromatin. Consequently, ALC1 deficiency enhances trapping of inhibited PARP1, which then impairs the binding of both nonhomologous end-joining and homologous recombination repair factors to DNA lesions. We also establish that ALC1 overexpression, a common feature in multiple tumor types, reduces the sensitivity of BRCA-deficient cells to PARP inhibitors. Together, we conclude that ALC1-dependent PARP1 mobilization is a key step underlying PARP inhibitor resistance.

scholarly journals Chromatin Architectural Factors as Safeguards against Excessive Supercoiling during DNA Replication

2020 ◽  
Vol 21 (12) ◽  
pp. 4504
Author(s):  
Syed Moiz Ahmed ◽  
Peter Dröge
Keyword(s):  
Dna Replication ◽  
Protein Complexes ◽  
Genome Replication ◽  
Relaxation Rates ◽  
Architectural Features ◽  
Genome Wide ◽  
New Findings ◽  
Accessory Factors ◽  
Novel Strategy ◽  
Supercoil Relaxation

Key DNA transactions, such as genome replication and transcription, rely on the speedy translocation of specialized protein complexes along a double-stranded, right-handed helical template. Physical tethering of these molecular machines during translocation, in conjunction with their internal architectural features, generates DNA topological strain in the form of template supercoiling. It is known that the build-up of transient excessive supercoiling poses severe threats to genome function and stability and that highly specialized enzymes—the topoisomerases (TOP)—have evolved to mitigate these threats. Furthermore, due to their intracellular abundance and fast supercoil relaxation rates, it is generally assumed that these enzymes are sufficient in coping with genome-wide bursts of excessive supercoiling. However, the recent discoveries of chromatin architectural factors that play important accessory functions have cast reasonable doubts on this concept. Here, we reviewed the background of these new findings and described emerging models of how these accessory factors contribute to supercoil homeostasis. We focused on DNA replication and the generation of positive (+) supercoiling in front of replisomes, where two accessory factors—GapR and HMGA2—from pro- and eukaryotic cells, respectively, appear to play important roles as sinks for excessive (+) supercoiling by employing a combination of supercoil constrainment and activation of topoisomerases. Looking forward, we expect that additional factors will be identified in the future as part of an expanding cellular repertoire to cope with bursts of topological strain. Furthermore, identifying antagonists that target these accessory factors and work synergistically with clinically relevant topoisomerase inhibitors could become an interesting novel strategy, leading to improved treatment outcomes.

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