Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

Bianco, Julien N; Poli, Jérôme; Saksouk, Julie; Bacal, Julien; Silva, Maria João; Yoshida, Kazumasa; Lin, Yi-Ching; Tourrière, Hélène; Lengronne, Armelle; Pasero, Philippe

doi:10.1016/j.ymeth.2012.04.007

Cited by 91 publications

(91 citation statements)

References 59 publications

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“…DNA combing was performed as described (72). BrdU was detected with a rat monoclonal antibody (AbCys; clone BU1/75) and goat anti-rat coupled to Alexa 488 (Invitrogen).…”

Section: Analysis Of Mcm4 Phosphorylationmentioning

confidence: 99%

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

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

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Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.

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“…DNA combing was performed as described (72). BrdU was detected with a rat monoclonal antibody (AbCys; clone BU1/75) and goat anti-rat coupled to Alexa 488 (Invitrogen).…”

Section: Analysis Of Mcm4 Phosphorylationmentioning

confidence: 99%

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

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

Self Cite

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“…DNA combing was performed as described (Bianco et al 2012) using a mouse monoclonal anti-ssDNA (Chemicon, and a rat monoclonal anti-BrdU (Abcys, clone BU1/75). Images were recorded on a Leica DM6000 microscope equipped with a CoolSNAP HQ CCD camera (Roper Scientific) and were processed as described (Bianco et al 2012).…”

Section: Dna Combingmentioning

confidence: 99%

“…Images were recorded on a Leica DM6000 microscope equipped with a CoolSNAP HQ CCD camera (Roper Scientific) and were processed as described (Bianco et al 2012).…”

Section: Dna Combingmentioning

confidence: 99%

Mec1, INO80, and the PAF1 complex cooperate to limit transcription replication conflicts through RNAPII removal during replication stress

et al. 2016

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Little is known about how cells ensure DNA replication in the face of RNA polymerase II (RNAPII)-mediated transcription, especially under conditions of replicative stress. Here we present genetic and proteomic analyses from budding yeast that uncover links between the DNA replication checkpoint sensor Mec1-Ddc2 (ATR-ATRIP), the chromatin remodeling complex INO80C (INO80 complex), and the transcription complex PAF1C (PAF1 complex). We found that a subset of chromatin-bound RNAPII is degraded in a manner dependent on Mec1, INO80, and PAF1 complexes in cells exposed to hydroxyurea (HU). On HU, Mec1 triggers the efficient removal of PAF1C and RNAPII from transcribed genes near early firing origins. Failure to evict RNAPII correlates inversely with recovery from replication stress: paf1Δ cells, like ino80 and mec1 mutants, fail to restart forks efficiently after stalling. Our data reveal unexpected synergies between INO80C, Mec1, and PAF1C in the maintenance of genome integrity and suggest a mechanism of RNAPII degradation that reduces transcription-replication fork collision.

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“…This procedure is suitable for measuring replication fork rates and replication origin usage. The protocol (Steps 23-66) is adapted from the procedures of the Schwob and Pasero laboratories (Lengronne et al 2001;Versini et al 2003;Schwob et al 2009;Bianco et al 2012). It is also amenable to more complicated double-labeling procedures involving the sequential addition of IdU and CldU to measure replication fork stalling and fork asymmetry.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Replicating Yeast Chromosomes by DNA Combing

Gallo¹,

Wang²,

Yip³

et al. 2016

Cold Spring Harb Protoc

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Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replication fork progression in the budding yeast Saccharomyces cerevisiae. In contrast to traditional flow cytometry, microarray, or sequencing techniques, which provide population-level data, DNA combing provides DNA replication profiles of individual molecules. DNA combing uses yeast strains that express human thymidine kinase, which facilitates the incorporation of thymidine analogs into nascent DNA. First, DNA is isolated and stretched uniformly onto silanized glass coverslips. Following immunodetection with antibodies that recognize the thymidine analog and the DNA, the DNA fibers are imaged using a fluorescence microscope. Finally, the lengths of newly replicated DNA tracks are measured and converted to base pairs, allowing calculations of the speed of the replication fork and of interorigin distances. DNA combing can be applied to monitor replication defects caused by gene mutations or by chemical agents that induce replication stress. Here, we present a methodology for studying replicating yeast chromosomes by molecular DNA combing. We begin with procedures for the preparation of silanized coverslips and for assembly of a DNA combing machine (DCM) and conclude by presenting a detailed protocol for molecular DNA combing in yeast. MATERIALS

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Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

Cited by 91 publications

References 59 publications

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Mec1, INO80, and the PAF1 complex cooperate to limit transcription replication conflicts through RNAPII removal during replication stress

Analysis of Replicating Yeast Chromosomes by DNA Combing

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