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Molecular and Cellular Biology, February 2006, p. 1051-1062, Vol. 26, No. 3
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.3.1051-1062.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Chinese Hamster Dihydrofolate Reductase Replication Origin Decision Point Follows Activation of Transcription and Suppresses Initiation of Replication within Transcription Units

Takayo Sasaki, Sunita Ramanathan,^, Yukiko Okuno,^, Chiharu Kumagai, Seemab S. Shaikh, and David M. Gilbert^*

Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams St., Syracuse, New York 13210

Received 7 September 2005/ Returned for modification 5 November 2005/ Accepted 15 November 2005

Chinese hamster ovary (CHO) cells select specific replication origin sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G₁ phase, the origin decision point (ODP). Origin selection is sensitive to transcription but not protein synthesis inhibitors, implicating a pretranslational role for transcription in origin specification. We have constructed a DNA array covering 121 kb surrounding the DHFR locus, to comprehensively investigate replication initiation and transcription in this region. When nuclei isolated within the first 3 h of G₁ phase were stimulated to initiate replication in Xenopus egg extracts, replication initiated without any detectable preference for specific sites. At the ODP, initiation became suppressed from within the Msh3, DHFR, and 2BE2121 transcription units. Active transcription was mostly confined to these transcription units, and inhibition of transcription by alpha-amanitin resulted in the initiation of replication within transcription units, indicating that transcription is necessary to limit initiation events to the intergenic region. However, the resumption of DHFR transcription after mitosis took place prior to the ODP and so is not on its own sufficient to suppress initiation of replication. Together, these results demonstrate a remarkable flexibility in sequence selection for initiating replication and implicate transcription as one important component of origin specification at the ODP.

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* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. Phone: (315) 464-8723. Fax: (315) 464-8750. E-mail: gilbertd{at}upstate. edu.

These authors contributed equally.

Present address: Roswell Park Memorial Institute, Molecular and Cellular Biology, 666 Elm St., Buffalo, NY 14263.

Present address: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

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Molecular and Cellular Biology, February 2006, p. 1051-1062, Vol. 26, No. 3
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.3.1051-1062.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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