Interplay between Sae2 and Rif2 in the regulation of Mre11-Rad50 activities at DNA ends

Abstract Background The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation. Results In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming. Conclusions We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.

Download Full-text

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Nature Communications ◽

10.1038/s41467-021-23015-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Elisa Oberbeckmann ◽

Vanessa Niebauer ◽

Shinya Watanabe ◽

Lucas Farnung ◽

Manuela Moldt ◽

...

Keyword(s):

Phased Arrays ◽

Stable Steady State ◽

Reference Sites ◽

Chromatin Remodelers ◽

Sliding Direction ◽

Nucleosome Dynamics ◽

Genome Wide ◽

Sequence Elements ◽

Nucleosome Array ◽

Dna Ends

AbstractArrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ‘ruler’ that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

Download Full-text

Telomeric and Sub-Telomeric Structure and Implications in Fungal Opportunistic Pathogens

Microorganisms ◽

10.3390/microorganisms9071405 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1405

Author(s):

Raffaella Diotti ◽

Michelle Esposito ◽

Chang Hui Shen

Keyword(s):

Fungal Pathogens ◽

Functional Abilities ◽

Opportunistic Pathogens ◽

Telomeric Sequences ◽

Coding Regions ◽

Telomeric Structure ◽

Linear Chromosomes ◽

Dna Ends ◽

Telomere Structure

Telomeres are long non-coding regions found at the ends of eukaryotic linear chromosomes. Although they have traditionally been associated with the protection of linear DNA ends to avoid gene losses during each round of DNA replication, recent studies have demonstrated that the role of these sequences and their adjacent regions go beyond just protecting chromosomal ends. Regions nearby to telomeric sequences have now been identified as having increased variability in the form of duplications and rearrangements that result in new functional abilities and biodiversity. Furthermore, unique fungal telomeric and chromatin structures have now extended clinical capabilities and understanding of pathogenicity levels. In this review, telomere structure, as well as functional implications, will be examined in opportunistic fungal pathogens, including Aspergillus fumigatus, Candida albicans, Candida glabrata, and Pneumocystis jirovecii.

Download Full-text

Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

Biochemistry ◽

10.1021/bi100503w ◽

2010 ◽

Vol 49 (29) ◽

pp. 6165-6176 ◽

Cited By ~ 63

Author(s):

Elizabeth Cotner-Gohara ◽

In-Kwon Kim ◽

Michal Hammel ◽

John A. Tainer ◽

Alan E. Tomkinson ◽

...

Keyword(s):

Bound States ◽

Dna Ligase ◽

Dynamic Switching ◽

Human Dna ◽

Dna Ends ◽

Dna Ligase Iii

Download Full-text

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

The Journal of Cell Biology ◽

10.1083/jcb.200608077 ◽

2007 ◽

Vol 177 (2) ◽

pp. 219-229 ◽

Cited By ~ 256

Author(s):

Naoya Uematsu ◽

Eric Weterings ◽

Ken-ichi Yano ◽

Keiko Morotomi-Yano ◽

Burkhard Jakob ◽

...

Keyword(s):

Kinase Activity ◽

Laser System ◽

Double Strand Breaks ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

Download Full-text

The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

Nucleic Acids Research ◽

10.1093/nar/gkp695 ◽

2009 ◽

Vol 37 (19) ◽

pp. 6389-6399 ◽

Cited By ~ 14

Author(s):

Sreerupa Ray ◽

Anne Grove

Keyword(s):

Chromatin Remodeling ◽

High Mobility ◽

High Mobility Group ◽

High Mobility Group Protein ◽

Chromatin Remodeling Complex ◽

A Subunit ◽

Group Protein ◽

Dna Ends

Download Full-text

How damaged is the biologically active subpopulation of transfected DNA?

Molecular and Cellular Biology ◽

10.1128/mcb.4.3.387-398.1984 ◽

1984 ◽

Vol 4 (3) ◽

pp. 387-398

Author(s):

C T Wake ◽

T Gudewicz ◽

T Porter ◽

A White ◽

J H Wilson

Keyword(s):

Simian Virus 40 ◽

Simian Virus ◽

Double Strand Breaks ◽

Biologically Active ◽

Exact Nature ◽

Dna Molecules ◽

Base Pairs ◽

Strand Breaks ◽

Dna Ends ◽

Prevalent Form

Relatively little is known about the damage suffered by transfected DNA molecules during their journey from outside the cell into the nucleus. To follow selectively the minor subpopulation that completes this journey, we devised a genetic approach using simian virus 40 DNA transfected with DEAE-dextran. We investigated this active subpopulation in three ways: (i) by assaying reciprocal pairs of mutant linear dimers which differed only in the arrangement of two mutant genomes; (ii) by assaying a series of wild-type oligomers which ranged from 1.1 to 2.0 simian virus 40 genomes in length; and (iii) by assaying linear monomers of simian virus 40 which were cleaved within a nonessential region to leave either sticky, blunt, or mismatched ends. We conclude from these studies that transfected DNA molecules in the active subpopulation are moderately damaged by fragmentation and modification of ends. As a whole, the active subpopulation suffers about one break per 5 to 15 kilobases, and about 15 to 20% of the molecules have one or both ends modified. Our analysis of fragmentation is consistent with the random introduction of double-strand breaks, whose cause and exact nature are unknown. Our analysis of end modification indicated that the most prevalent form of damage involved deletion or addition of less than 25 base pairs. In addition we demonstrated directly that the efficiencies of joining sticky, blunt, or mismatched ends are identical, verifying the apparent ability of cells to join nearly any two DNA ends and suggesting that the efficiency of joining approaches 100%. The design of these experiments ensured that the detected damage preceded viral replication and thus should be common to all DNAs transfected with DEAE-dextran and not specific for viral DNA. These measurements of damage within transfected DNA have important consequences for studies of homologous and nonhomologous recombination in somatic cells as is discussed.

Download Full-text

Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00156-17 ◽

2017 ◽

Vol 37 (24) ◽

Cited By ~ 8

Author(s):

Sucheta Arora ◽

Rajashree A. Deshpande ◽

Martin Budd ◽

Judy Campbell ◽

America Revere ◽

...

Keyword(s):

Nuclease Activity ◽

Double Strand Breaks ◽

Endonuclease Activity ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Content Type ◽

Strand Breaks ◽

Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

Download Full-text