Mechanisms for initiating cellular DNA replication

Bleichert, Franziska; Botchan, Michael R.; Berger, James M.

doi:10.1126/science.aah6317

Cited by 196 publications

(203 citation statements)

References 322 publications

(332 reference statements)

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“…Metaplots were generated using Prism Graphpad. 18, 19, 20, 22, 23, 25, 27, 28, 32 09, 10, 12, 15, 16, 17, 24, 26, 31, 33, 36 04, 06, 07, 11,13,14,21,29,30,34,35…”

Section: Meta-analysismentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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Once every cell cycle, DNA replication takes place to allow cells to duplicate their genome and segregate the two resulting copies into offspring cells. In eukaryotes, the number of DNA replication initiation loci, termed origins, is proportional to chromosome size. However, previous studies have suggested that in Leishmania, a group of single-celled eukaryotic parasites, DNA replication starts from just a single origin per chromosome, which is predicted to be insufficient to secure complete genome duplication within S phase.Here, we show that the paucity of origins activated in early S phase is balanced by DNA synthesis activity outside S phase. Simultaneous recruitment of acetylated histone H3 (AcH3), modified base J and the kinetochore factor KKT1 is exclusively found at the origins used in early S phase, while subtelomeric DNA replication can only be linked to AcH3 and displays persistent activity through the cell cycle, including in G2/M and G1 phases. We also show that subtelomeric DNA replication, unlike replication from the previously mapped origins, is sensitive to hydroxyurea and dependent on subunits of the 9-1-1 complex.Our work indicates that Leishmania genome transmission relies on an unconventional DNA replication programme, which may have implications for genome stability in this important parasite. sequence read depth in the former relative to the latter. As result, DNA synthesis very late in S-phase, or that occurs outside of S-phase, would escape detection. To test this, we modified the MFA-seq approach in order that DNA content enrichment could be calculated in replicating cells relative to naturally occurring non-replicative cells. Mapping origins of replication by calculating DNA enrichment in exponentially growing cells versus cells in a stationary state has been used successfully in bacteria (42) and yeast (43). Thus, we reasoned that L. major in stationary phase could also serve as a non-replicative control for normalization in MFA-seq analysis compared with cells that are growing exponentially. To test this, we used flow cytometry to compare DNA content of exponentially growing and stationary phase cells and, in addition, compared the capacity of the two cell populations to incorporate the thymidine analogue 5-ethynyl-2'deoxyuridine (EdU; Fig. 1A,B). Flow cytometry showed that the proportion of cells in S phase (with DNA content between 1C and 2C) was substantially lower in a population of stationary cells compared with exponentially growing cells (Fig. 1A). Concomitantly, stationary phase cells, unlike exponentially growing cells, failed to incorporate EdU, even upon relatively long periods of incubation ( Fig. 1B). Thus, L. major cells in stationary phase do not perform any detectable DNA synthesis and were therefore deemed suitable to be used as the non-replicative sample in MFA-seq analysis.Next, we compared MFA-seq profiles using read depth ratios from FACS-sorted early S (ES) and G2 cells, and from exponentially growing cells (EXP) and stationary (STA) cells (Fig. 1C). As reported ...

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“…Metaplots were generated using Prism Graphpad. 18, 19, 20, 22, 23, 25, 27, 28, 32 09, 10, 12, 15, 16, 17, 24, 26, 31, 33, 36 04, 06, 07, 11,13,14,21,29,30,34,35…”

Section: Meta-analysismentioning

confidence: 99%

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Damasceno

Marques

Beraldi

et al. 2019

Preprint

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“…All cells utilize factors that assemble two helicases onto DNA for bidirectional replication forks. In Escherichia coli the DnaA origin binding protein is primarily responsible for the initial opening of double-strand (ds) DNA, upon which two DnaB hexameric helicase rings are assembled onto opposite strands of the singlestranded (ss) DNA bubble (reviewed in (Bleichert et al, 2017;O'Donnell et al, 2013)). Unlike bacteria, studies in Saccharomyces cerevisiae reveal that eukaryotic helicases are assembled on dsDNA.…”

Section: Introductionmentioning

confidence: 99%

Supporting evidence for DNA shearing as a mechanism for origin unwinding in eukaryotes

Langston

Georgescu

O’Donnell

2019

Preprint

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Our earlier study demonstrated that head-to-head CMGs that encircle duplex DNA and track inward at origins melt double-strand (ds) DNA while encircling the duplex by pulling on opposite strands and shearing DNA apart (Langston and O'Donnell (2019) eLife 9, e46515). We show here that increasing the methylphosphonate neutral DNA from 10 nucleotides in the previous report, to 20 nucleotides, reveals that CMG encircling duplex DNA only interacts with the tracking strand compared to the non-tracking strand. This significantly enhances support that CMG tracks on duplex DNA by binding only one strand. Furthermore, EMSA assays using AMPPNP to load CMG onto DNA shows a stoichiometry of only 2 CMGs on an origin mimic DNA, containing a 150 bp duplex with two 3 prime single-strand (ss) tails, one on each end, enabling assay of dsDNA unwinding by a shearing force produced by only two head-to-head CMGs. The use of non-hydrolysable AMPPNP enabled a preincubation for CMG binding the two 3 prime tailed origin mimic DNA, and gave robust unwinding of dsDNA by head-tohead CMG-Mcm10's. With this precedent, it is possible to envision that the cell may utilize opposing dsDNA motors to unwind DNA for other types of DNA transactions besides origin unwinding.

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“…The most widespread molecular systems based on sequence recognition involve dedicated nucleic acid-binding proteins (1,2). In particular, promoter recognition by transcription factors and recognition of chromosomal replication origins by initiation proteins are fundamental, universal processes central to normal cell function (3,4). Additionally, recognition of nucleic acids by proteins is the basis of self vs. nonself discrimination that is essential for defense functions, such as restriction modification in prokaryotes (5,6).…”

mentioning

confidence: 99%

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Peters

Makarova

Shmakov

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

244

267

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A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNAguided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.CRISPR-Cas systems | Tn7 transposon | transposition strategy | crRNA guide | target-site selection

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Mechanisms for initiating cellular DNA replication

Cited by 196 publications

References 322 publications

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Genome duplication inLeishmania majorrelies on DNA replication outside S phase

Supporting evidence for DNA shearing as a mechanism for origin unwinding in eukaryotes

Recruitment of CRISPR-Cas systems by Tn7-like transposons

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