Dynamics of plant DNA replication based on PCNA visualization.

Ryohei Yokoyama,Takeshi Hirakawa,Seri Hayashi,Takuya Sakamoto,Sachihiro Matsunaga

doi:10.1038/srep29657

Ryohei Yokoyama, Takeshi Hirakawa + Show 3 more

Open Access

https://doi.org/10.1038/srep29657

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Journal: Scientific reports	Publication Date: Jul 15, 2016
Citations: 39	License type: CC BY 4.0

Affiliation: Tokyo University of Science

Abstract

DNA replication is an essential process for the copying of genomic information in living organisms. Imaging of DNA replication in tissues and organs is mainly performed using fixed cells after incorporation of thymidine analogs. To establish a useful marker line to measure the duration of DNA replication and analyze the dynamics of DNA replication, we focused on the proliferating cell nuclear antigen (PCNA), which functions as a DNA sliding clamp for replicative DNA polymerases and is an essential component of replisomes. In this study we produced an Arabidopsis thaliana line expressing PCNA1 fused with the green fluorescent protein under the control of its own promoter (pAtPCNA1::AtPCNA1-EGFP). The duration of the S phase measured using the expression line was consistent with that measured after incorporation of a thymidine analog. Live cell imaging revealed that three distinct nuclear localization patterns (whole, dotted, and speckled) were sequentially observable. These whole, dotted, and speckled patterns of subnuclear AtPCNA1 signals were indicative of the G1 or G2 phase, early S phase and late S phase, respectively. The results indicate that the pAtPCNA1::AtPCNA1-EGFP line is a useful marker line for visualization of S-phase progression in live plant organs.

Highlights

We observed AtPCNA1sGFP signals in nuclei of the epidermal cells of leaves, trichomes, hypocotyls, the meristematic, elongation and differentiation zones of roots, and lateral root primordia (Fig. 1a–g). These results indicated that AtPCNA1 is expressed in nuclei of diverse tissues of A. thaliana
An inhibitor of DNA polymerase α, which catalyzes the synthesis of template RNA required for initiation of DNA replication[20], the dotted and speckled patterns of AtPCNA1 signals were observed at high frequency (Fig. 5a,b)
The whole pattern of AtPCNA1 signals does not exhibit the S phase, EdU signals were weakly detected in the nuclei that showed the whole pattern of AtPCNA1 signals (Type I) (Fig. 4c)

Summary

Introduction

DAPI-positive subnuclear regions, chromocenters, were detected in the nuclei with the whole pattern of AtPCNA1 signals, but were not observed in the case of the speckled signal pattern (Fig. 2f). To investigate how the three patterns of AtPCNA1 signals sequentially changed through the cell cycle, we performed time-lapse imaging of AtPCNA1-sGFP roots in the meristematic zone. When the duration of each pattern of AtPCNA1-sGFP signals was measured, the duration of the dotted and speckled patterns was approximately 60 and 80 min, respectively (Fig. 3b), indicating that the subnuclear localization of AtPCNA1 dynamically changed in the root meristematic zone.

Results

Conclusion