Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts

Yuta Nakazaki; Takashi Tsuyama; Masayuki Seki; Mikiko Takahashi; Takemi Enomoto; Shusuke Tada

doi:10.1016/j.bbrc.2016.01.028

Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts

Biochem Biophys Res Commun. 2016 Feb 5;470(2):405-410. doi: 10.1016/j.bbrc.2016.01.028. Epub 2016 Jan 8.

Authors

Yuta Nakazaki¹, Takashi Tsuyama², Masayuki Seki³, Mikiko Takahashi¹, Takemi Enomoto⁴, Shusuke Tada⁵

Affiliations

¹ Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo, Japan.
² Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba, Japan.
³ Department of Biochemistry, Tohoku Pharmaceutical University, Sendai, Miyagi, Japan.
⁴ Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Nishitokyo-shi, Tokyo, Japan.
⁵ Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo, Japan; Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba, Japan. Electronic address: s.tada@phar.toho-u.ac.jp.

PMID: 26773501
DOI: 10.1016/j.bbrc.2016.01.028

Abstract

Cdt1 is a protein essential for initiation of DNA replication; it recruits MCM helicase, a core component of the replicative DNA helicase, onto replication origins. In our previous study, we showed that addition of excess Cdt1 inhibits nascent strand elongation during DNA replication in Xenopus egg extracts. In the present study, we investigated the mechanism behind the inhibitory effect of Cdt1. We found that addition of recombinant Cdt1 inhibited nascent DNA synthesis in a reinitiation-independent manner. To identify the mechanism by which Cdt1 inhibits nascent strand elongation, the effect of Cdt1 on loading of Mcm4 and Rpa70 onto chromatin was examined. The results showed that Cdt1 suppressed the excessive Rpa70 binding caused by extensive, aphidicolin-induced DNA unwinding; this unwinding occurs between stalled DNA polymerases and advancing replication forks. These findings suggested that excess Cdt1 suppressed the progression of replication forks.

Keywords: Cdt1; DNA replication; MCM helicase; Xenopus egg extracts.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Cycle Proteins / genetics*
DNA Replication / genetics*
Ovum / physiology*
Transcription Elongation, Genetic / physiology*
Xenopus laevis

Substances

CDT1 protein, human
Cell Cycle Proteins